Hepatitis G virus and molecular cloning thereof

ABSTRACT

Polypeptide antigens are disclosed which are immunoreactive with sera from individuals having a non-A, non-B, non-C, non-D, non-E Hepatitis, herein designated Hepatitis G Virus (HGV). Corresponding genomic-fragment clones containing polynucleotides encoding the open reading frame sequences for the antigenic polypeptides are taught. The antigens are useful in diagnostic methods for detecting the presence of HGV in test subjects. The antigens are also useful in vaccine and antibody preparations. In addition, the entire coding sequences of two HGV isolates are disclosed. Methods are presented for nucleic acid-based detection of HGV in samples and also methods for the isolation of further genomic sequences corresponding to HGV.

This application is a divisional of U.S. application Ser. No.08/444,733, filed May 19, 1995, which is a continuation-in-part of U.S.application Ser. No. 08/344,271, filed Nov. 23, 1994, abandoned, whichis a continuation-in-part of U.S. application Ser. No. 08/285,561, filedon Aug. 3, 1994, abandoned, which is a continuation-in-part of U.S.application Ser. No. 08/264,985 filed on May 20, 1994, abandoned, and isa continuation-in-part of U.S. patent application Ser. No. 08/389,886,filed Feb. 15, 1995 abandoned, herein incorporated by reference, whichis a continuation-in-part of 08/357,509, filed Dec. 16, 1994 abandoned,herein incorporated by reference, which is a continuation-in-part ofU.S. patent application Ser. No. 08/329,729, filed Oct. 26, 1994abandoned, herein incorporated by reference, which is acontinuation-in-part of U.S. patent application Ser. No. 08/285,558,filed Aug. 3, 1994 abandoned, and U.S. patent application Ser. No.08/285,543 abandoned, filed Aug. 3, 1994, herein incorporated byreference, which are continuations-in-part of U.S. patent applicationSer. No. 08/246,985, filed May 20, 1994 abandoned, herein incorporatedby reference.

FIELD OF INVENTION

This invention relates to nucleic acid, polypeptide, antigen, epitope,vaccine and antibody compositions related to a NonA/NonB/NonC/NonD/NonE(N-(ABCDE)) hepatitis-associated viral agent (HGV). The invention alsorelates to diagnostic and therapeutic methods.

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BACKGROUND OF THE INVENTION

Viral hepatitis resulting from a virus other than hepatitis A virus(HAV) and hepatitis B virus (HBV) has been referred to as non-A, non-Bhepatitis (NANBH). NANBH can be further defined based on the mode oftransmission of an individual type, for example, enteric versusparenteral.

One form of NANBH, known as enterically transmitted NANBH or ET-NANBH,is contracted predominantly in poor-sanitation areas where food anddrinking water have been contaminated by fecal matter. The molecularcloning of the causative agent, referred to as the hepatitis E virus(HEV), has recently been described (Reyes et al., 1990; Tam et al.).

A second form of NANB, known as parenterally transmitted NANBH, orPT-NANBH, is transmitted by parenteral routes, typically by exposure toblood or blood products. The rate of this hepatitis varied by (i)locale, (ii) whether ALT testing was done in blood banks, and (iii)elimination of high-risk patients for AIDS. Appoximately 10% oftransfusions caused PT-NANBH infection and about half of those went onto a chronic disease state (Dienstag). After implementation of anti-HCVtesting, HCV seroconversion per unit transfused was decreased to lessthan 1% among heart surgery patients (Alter).

Human plasma samples documented as having produced post-transfusionNANBH in human recipients have been used successfully to producePT-NANBH infection in chimpanzees (Bradley). RNA isolated from infectedchimpanzee plasma has been used to construct cDNA libraries in anexpression vector for immunoscreening with serum from human subjectswith chronic PT-NANBH infection. This procedure identified a PT-NANBHspecific cDNA clone and the viral sequence was then used as a probe toidentify a set of overlapping fragments making up 7,300 contiguousbasepairs of a PT-NANBH viral agent. The sequenced viral agent has beennamed the hepatitis C virus (HCV) (for example, the sequence of HCV ispresented in EPO patent application 88310922.5, filed 11/18/88). Thefull-length sequence (˜9,500 nt) of HCV is now available.

Primate transmission studies conducted at the Centers for DiseaseControl (CDC; Phoenix, Ariz., 1973-1975; 1978-1983) originally providedsubstantial evidence for the existence of multiple agents of non-A,non-B hepatitis (NANBH): the primary agents associated with the majorityof cases of NANBH are now recognized to be HCV and HEV (see above), forPT-NANBH and ET-NANBH, respectively. Later epidemiologic studiesconducted at the CDC (Atlanta, Ga., 1989-present) using both research(prototype) and commercial tests for anti-HCV antibody showed thatapproximately 20% of all community-acquired NANBH was also non-C.Further testing of these samples for the presence of HEV (Reyes, et al.,WO A 9115603 (Genelabs Inc.) Oct. 17, 1991) have indicated that thesecases of community-acquired non-A, non-B, non-C hepatitis were alsonon-E.

Liver biopsy specimens, sera and plasma of Sentinel County patients(study of Drs. Miriam Alter and Kris Krawczynski) also showed that manybona fide cases of NANBH were also non-C hepatitis (serologically and byReverse Transcriptase-Polymerase Chain Reaction (RT-PCR; Kawasaki, etal.; Wang, et al., 1990) negative for all markers of HCV infection)developed subsequently into chronic hepatitis with presentation ofchronic persistent hepatitis (CPH) or chronic active hepatitis (CAH)consistent with a viral infection.

SUMMARY OF THE INVENTION

The invention pertains to the characterization and isolation of a newlydiscovered NonA/NonB/NonC/NonD/--NonE (N--(ABCDE)) hepatitis-associatedviral agent, herein designated Hepatitis G Virus (HGV). Disclosed hereis a family of CDNA replicas of portions of HGV genome. Also disclosedare methods for the isolation and characterization of further HGVsequences and sequences of HGV variants.

The present invention includes HGV genomic polynucleotides, cDNAsthereto and complements thereof. With respect to polynucleotides, someaspects of the invention include: a purified Hepatitis G Virus genomicpolynucleotide; HGV derived RNA and DNA polynucleotides; recombinant HGVpolynucleotides; a recombinant polynucleotide making up a sequencederived from HGV or HGV variant cDNA or complementary sequences thereof;a recombinant polynucleotide encoding an epitope of HGV; a recombinantvector including any of the above recombinant polynucleotides, and ahost cell transformed with any of these vectors. Another aspect of theinvention is a polynucleotide probe for HGV and/or its variants.

Current studies on the nature of the genome of HGV, utilizing sequenceinformation to compare HGV to other viral sequences, suggest that HGV isa member of the Flaviviridae family of viruses.

Portions of the HGV-derived CDNA sequences are effective as probes toisolate variants of the virus which occur naturally, or to determine thepresence of virus in samples. These cDNAs also make availableHGV-encoded polypeptide sequences, including HGV-specific polypeptideantigens. These coding sequences allow the production of polypeptideswhich are useful as reagents in diagnostic tests and/or as components ofvaccines, or as standards. Further, it is possible to isolate andsequence other portions of the HGV genome by utilizing probes derivedfrom these cDNAs, therefore giving rise to additional probes andpolypeptides useful in the prophylactic, therapeutic and diagnosisapplications.

Other aspects of the invention include: a recombinant expression systemwhich incorporates an open reading frame (ORF) derived from HGV cDNA orcomplements thereof, wherein the ORF is linked operably to a controlsequence which is compatible with a desired host, a cell transformedwith the recombinant expression system, and a polypeptide produced bythe transformed cell.

Yet another aspect of the invention are purified HGV particles; apreparation of polypeptides from the purified HGV; a purified HGVpolypeptide; a purified HGV peptide; and a purified polypeptide whichcomprises an epitope immunologically identifiable with an epitopecontained in HGV or an HGV variant.

Included aspects of the invention are an HGV polypeptide; a recombinantpolypeptide consisting of a sequence derived from a HGV genome, HGV cDNAor complements thereof; a recombinant polypeptide made of an HGVepitope; and a fusion polypeptide comprised of an HGV polypeptide.

Both polyclonal and monoclonal antibodies directed against HGV epitopescontained within the polypeptide sequences are also useful astherapeutic agents, for diagnostic tests, for the isolation of the HGVagent from which these cDNAs derive, and for screening of antiviralagents.

Also included in the invention are a purified preparation of polyclonalantibodies directed against an HGV epitope; and monoclonal antibodiesdirected against HGV epitopes.

Some aspects of the invention pertaining to kits are those for:investigating samples for the presence of polynucleotides derived fromHGV which comprise a polynucleotide probe including a nucleotidesequence from HGV of approximately 8 or more nucleotides, in anappropriate container; analyzing samples for the presence of antibodiesdirected against an HGV antigen made up of a polypeptide which containsan HGV epitope present in the HGV antigen, in a suitable container; andanalyzing samples for the presence of HGV antigens made up of ananti-HGV antibody, in a suitable container.

Still other aspects of the invention include a polypeptide comprised ofan HGV epitope, which is attached to a solid substrate; and an antibodyto an HGV epitope, which is attached to a solid substrate.

Other aspects of the invention are: a technique for the production of anHGV polypeptide, which includes incubating host cells which aretransformed with an expression vector, containing a sequence encoding anHGV polypeptide, under conditions which allow expression of saidpolypeptide; and a polypeptide which has been produced by this method(containing, for example, an HGV epitope).

Also included in the invention are a method for the detection of HGVnucleic acids in samples comprising reacting nucleic acids of the samplewith a probe for an HGV polynucleotide, under conditions allowing thecreation of a polynucleotide duplex between the probe and the HGVnucleic acid from the sample; as well as detecting a polynucleotideduplex containing the probe. The invention includes the followinghybridization based detection methods: reporter labeling; polymerasechain reaction; self-sustained sequence replication; ligase chainreaction; and strand displacement amplification. Further, detectionmethods include signal amplification (e.g., branch-chained DNA probesand the Q-beta replicase method).

The invention also includes immunoassays, including an immunoassay fordetecting HGV, comprising the incubation of a sample (which is suspectedof being infected with HGV) with a probe antibody directed against anantigen/epitope of HGV, to be detected under conditions allowing theformation of an antigen-antibody complex; and detecting theantigen-antibody complex which contains the probe antibody. Animmunoassay for the detection of antibodies which are directed againstan HGV antigen comprising the incubation of a sample suspected ofcontaining HGV with a probe polypeptide including an epitope of HGV,under conditions that allow the formation of an antibody-antigencomplex; and distinguishing the antibody-antigen complex which containsthe probe antigen.

Also forming part of the invention are HGV vaccines, for the treatmentand/or prevention of HGV infection, comprising an immunogenic peptidecontaining an HGV epitope, or an inactivated preparation of HGV, or areduced preparation of HGV.

In still another aspect, the invention includes a tissue culture growncell, infected with HGV. In one embodiment, the tissue culture growncells are primate liver cells.

Another aspect of the invention is a method for producing antibodies toHGV, comprising administering to a test subject an immunogenicpolypeptide containing HGV epitopes in an adequate amount to elicit animmune response.

The present invention also includes an HGV mosaic polypeptide, where themosaic polypeptide contains at least two epitopes of HGV, and, where thepolypeptide substantially lacks amino acids normally intervening betweenthe epitopes in the native HGV coding sequence. Such mosaic polypeptidesare useful in the applications and methods discussed above.

The present invention further includes a random peptide epitope(mimitope) that mimics a natural HGV antigenic epitope during epitopepresentation. Such mimitopes are useful in the applications and methodsdiscussed above. Also included in the present invention is a method ofidentifying a random peptide HGV epitope. In the method, a library ofrandom peptide epitopes is generated or selected. The library iscontacted with an anti-HGV antibody. Mimitopes are identified that arespecifically immunoreactive with the antibody. Sera (containing anti-HGVantibodies) or antibodies generated by the methods of the presentinvention can be used. Random peptide libraries can, for example, bedisplayed on phage or generated as combinatorial libraries.

In another aspect, the present invention includes therapeutic compoundsand methods for the prevention and/or treatment of HGV infection.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: the relationship of the SEQ ID NO:14 open reading frame to the470-20-1 clone.

FIG. 2: shows an exemplary protein profile from gradient fractionseluted from a glutathione affinity column.

FIG. 3: shows an exemplary Sodium dodecyl sulfate polyacrylamide gelelectrophoresis analysis of fraction samples from FIG. 2.

FIG. 4A: shows an exemplary protein profile from gradient fractionseluted from an anion exchange column.

FIGS. 4B and 4C: show exemplary Sodium dodecyl sulfate polyacrylamidegel electrophoresis analysis of fraction samples from FIG. 4A.

FIGS. 5A and 5B: amino acid alignments of HGV with two other members ofFlaviviridae family--Hog Cholera Virus and Hepatitis C Virus.

FIG. 6 shows a map of a portion of the vector pGEX-Hisb-GE3-2, abacterial expression plasmid carrying an HGV epitope.

FIGS. 7A to 7D show the results of Western blot analysis of the purifiedHGV GE3-2 protein.

FIGS. 8A to 8D show the results of Western blot analysis of the purifiedHGV Y5-10 antigen.

FIGS. 9A to 9D show the results of Western blot analysis of thefollowing antigens: Y5-5, GE3-2 and Y5-10.

FIGS. 10A to 11F show the results of Western blot analysis of antigensGE-NS2b and GE-NS5a.

FIG. 11 presents a Kyte-Doolittle hydrophobicity plot of the codingsequence of HGV.

FIG. 12 shows the results of Western blot analysis of HGV pET cloneswith anti-T7.Tag monoclonal antibody.

FIGS. 13A to 13D show the results of Western blot analysis of HGV pETclone GE-NS5b. FIG. 13E shows a corresponding coomassie stained gel.

FIGS. 14A to 14C show the results of Western blot analysis of HGV pETclone GE-E2. FIG. 14D shows a corresponding coomassie stained gel.

FIGS. 15A to 15C show the results of Western blot analysis of HGV pETclone GE-NS5b. FIG. 15D shows a corresponding coomassie stained gel.

FIG. 16 shows a schematic representation of the coding regions of HGV.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

The terms defined below have the following meaning herein:

1. "nonA/nonB/nonC/nonD/nonE hepatitis viral agent {N-(ABCDE)}", hereinprovisionally designated HGV, means a virus, virus type, or virus classwhich (i) is transmissible in some primates, including, mystax,chimpanzees or humans as characterized by elevated serum alanineamino-transferase levels in an infected primate (ii) is serologicallydistinct from hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis D virus, and hepatitis E (HEV)(although HGV may co-infect a subject with these viruses), and (iii) isa member of the virus family Flaviviridae.

2. "HGV variants" are defined as viral isolates that have at least about40%, preferably 55% or 65%, or more preferably 80% global sequencehomology, that is, sequence identity over a length of the viral genomepolynucleotide sequence, to the HGV polynucleotide sequences disclosedherein (e.g., SEQ ID NO:14).

"Sequence homology" is determined essentially as follows. Twopolynucleotide sequences of similar length (preferably, the entire viralgenome) are considered to be homologous to one another, if, when theyare aligned using the ALIGN program, over 40%, preferably 55% or 65%, ormore preferably 80% of the nucleic acids in the highest scoringalignment are identically aligned using a ktup of 1, the defaultparameters and the default PAM matrix.

The ALIGN program is found in the FASTA version 1.7 suite of sequencecomparison programs (Pearson, et al., 1988; Pearson, 1990; programavailable from William R. Pearson, Department of Biological Chemistry,Box 440, Jordan Hall, Charlottesville, Va.).

In determining whether two viruses are "highly homologous" to eachother, the complete sequence of all the viral proteins (or thepolyprotein) for one virus are optimally, globally aligned with theviral proteins or polyprotein of the other virus using the ALIGN programof the above suite using a ktup of 1, the default parameters and thedefault PAM matrix. Regions of dissimilarity or similarity are notexcluded from the analysis. Differences in lengths between the twosequences are considered as mismatches. Alternatively, viral structuralprotein regions are typically used to determine relatedness betweenviral isolates. Highly homologous viruses have over 40%, or preferably55% or 65%, or more preferably 80% global polypeptide sequence identity.

3. Two nucleic acid fragments are considered to be "selectivelyhybridizable" to an HGV polynucleotide, if they are capable ofspecifically hybridizing to HGV or a variant thereof (e.g., a probe thathybridizes to HGV nucleic acid but not to polynucleotides from othermembers of the virus family Flaviviridae) or specifically priming apolymerase chain reaction: (i) under typical hybridization and washconditions, as described, for example, in Maniatis, et al., pages320-328, and 382-389, (ii) using reduced stringency wash conditions thatallow at most about 25-30% basepair mismatches, for example: 2×SSC, 0.1%SDS, room temperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 37°C. once, 30 minutes; then 2×SSC room temperature twice, 10 minutes each,or (iii) selecting primers for use in typical polymerase chain reactions(PCR) under standard conditions (for example, in Saiki, R. K, et al.),which result in specific amplification of sequences of HGV or itsvariants.

Preferably, highly homologous nucleic acid strands contain less than20-30% basepair mismatches, even more preferably less than 5-20%basepair mismatches. These degrees of homology can be selected by usingwash conditions of appropriate stringency for identification of clonesfrom gene libraries (or other sources of genetic material), as is wellknown in the art.

4. An "HGV polynucleotide," as used herein, is defined as follows. Forpolynucleotides greater than about 100 nucleotides, HGV polynucleotidesencompass polynucleotide sequences encoded by HGV variants andhomologous sequences as defined in "2" above. For polynucleotides lessthan about 100 nucleotides in length, HGV polynucleotide encompassessequences that selectively hybridizes to sequences of HGV or itsvariants. Further, HGV polynucleotides include polynucleotides encodingHGV polypeptides (see below).

The term "polynucleotide" as used herein refers to a polymeric moleculehaving a backbone that supports bases capable of hydrogen bonding totypical nucleic acids, where the polymer backbone presents the bases ina manner to permit such hydrogen bonding in a sequence specific fashionbetween the polymeric molecule and a typically nucleic acid (e.g.,single-stranded DNA). Such bases are typically inosine, adenosine,guanosine, cytosine, uracil and thymidine. Numerous polynucleotidemodifications are known in the art, for example, labels, methylation,and substitution of one or more of the naturally occurring nucleotideswith an analog.

Polymeric molecules include double and single stranded RNA and DNA, andbackbone modifications thereof, for example, methylphosphonate linkages.Further, such polymeric molecules include alternative polymer backbonestructures such as, but not limited to, polyvinyl backbones (Pitha,1970a/b), morpholino backbones (Summerton, et al., 1992, 1993). Avariety of other charged and uncharged polynucleotide analogs have beenreported. Numerous backbone modifications are known in the art,including, but not limited to, uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, and carbamates) andcharged linkages (e.g., phosphorothioates and phosphorodithioates). Inaddition linkages may contain the following exemplary modifications:pendant moieties, such as, proteins (including, for example, nucleases,toxins, antibodies, signal peptides and poly-L-lysine); intercalators(e.g., acridine and psoralen), chelators (e.g., metals, radioactivemetals, boron and oxidative metals), alkylators, and other modifiedlinkages (e.g., alpha anomeric nucleic acids).

5. An "HGV polypeptide" is defined herein as any polypeptide homologousto an HGV polypeptide. "Homology," as used herein, is defined asfollows. In one embodiment, a polypeptide is homologous to an HGVpolypeptide if it is encoded by nucleic acid that selectively hybridizesto sequences of HGV or its variants.

In another embodiment, a polypeptide is homologous to an HGV polypeptideif it is encoded by HGV or its variants, as defined above, polypeptidesof this group are typically larger than 15, preferable 25, or morepreferable 35, contiguous amino acids. Further, for polypeptides longerthan about 60 amino acids, sequence comparisons for the purpose ofdetermining "polypeptide homology" are performed using the localalignment program LALIGN. The polypeptide sequence, is compared againstthe HGV amino acid sequence or any of its variants, as defined above,using the LALIGN program with a ktup of 1, default parameters and thedefault PAM.

Any polypeptide (typically a polypeptide not specifically immunoreactivewith HGV antibodies) with an optimal alignment longer than 60 aminoacids and greater than 60%, preferably 70%, or more preferably 80% ofidentically aligned amino acids is considered to be a "homologouspolypeptide." The LALIGN program is found in the FASTA version 1.7 suiteof sequence comparison programs (Pearson, et al., 1988; Pearson, 1990;program available from William R. Pearson, Department of BiologicalChemistry, Box 440, Jordan Hall, Charlottesville, Va.).

6. A polynucleotide is "derived from" HGV if it has the same orsubstantially the same basepair sequence as a region of an HGV genome,CDNA of HGV or complements thereof, or if it displays homology as notedunder "2", "3" or "4" above.

A polypeptide or polypeptide "fragment" is "derived from" HGV if it is(i) encoded by an open reading frame of an HGV polynucleotide, or (ii)displays homology to HGV polypeptides as noted under "2" and "5" above,or (iii) is specifically immunoreactive with HGV positive sera.

7. "Substantially isolated" and "purified" are used in several contextsand typically refer to at least partial purification of an HGV virusparticle, component (e.g., polynucleotide or polypeptide), or relatedcompound (e.g., anti-HGV antibodies) away from unrelated orcontaminating components (e.g., serum cells, proteins, non-HGVpolynucleotides and non-anti-HGV antibodies). Methods and procedures forthe isolation or purification of compounds or components of interest aredescribed below (e.g., affinity purification of fusion proteins andrecombinant production of HGV polypeptides).

8. In the context of the present invention, the phrase "nucleic acidsequences," when referring to sequences which encode a protein,polypeptide, or peptide, is meant to include degenerative nucleic acidsequences which encode homologous protein, polypeptide or peptidesequences as well as the disclosed sequence.

9. An "epitope" is the antigenic determinant defined as the specificportion of an antigen with which the antigen binding portion of aspecific antibody interacts.

10. An antigen or epitope is "specifically immunoreactive" with HGVpositive sera when the epitope/antigen binds to antibodies present inthe HGV infected sera but does not bind to antibodies present in themajority (greater than about 90%, preferably greater than 95%) of serafrom individuals who are not or have not been infected with HGV."Specifically immunoreactive" antigens or epitopes may also beimmunoreactive with monoclonal or polyclonal antibodies generatedagainst specific HGV epitopes or antigens.

An antibody or antibody composition (e.g., polyclonal antibodies) is"specifically immunoreactive" with HGV when the antibody or antibodycomposition is immunoreactive with an HGV antigen but not with HAV, HBV,HCV, HDV or HEV antigens. Further, "specifically immunoreactiveantibodies" are not immunoreactive with antigens typically present innormal sera obtained from subjects not infected with or exposed to HGV,HAV, HBV, HCV, HDV or HEV.

II. N- (ABCDE) SERA.

Availability of a serologic test for anti-HCV and the development of anRT-PCR assay for HCV-RNA (Kawasaki, et al.; Wang, et al., 1990) allowedthe identification of several cases of both post-transfusion andcommunity acquired non-HCV hepatitis. The human hepatitis case, PNF2161, was originally identified as having NANB hepatitis (NANBH) throughthe Sentinel Counties Study of community acquired hepatitis, sponsoredby the Centers for Disease Control and Prevention (Alter, et al.,1989b). PNF 2161 was a sample obtained from an elderly Caucasian malepatient who developed acute hepatitis approximately 8 weeks following ablood transfusion, with a peak serum ALT level of 1141 IU (normal, <45IU). Following resolution of the episode of acute hepatitis, he hadfluctuating, but persistently elevated ALT levels over the next sevenyears, consistent with chronic hepatitis, although histopathologicconfirmation of this diagnosis was not obtained.

The plasma specimen used to clone HGV (as described herein) was obtainedin June 1989, approximately 4^(1/) 2 years following the episode ofacute hepatitis, and cryo-preserved. Patient PNF 2161 was initiallybelieved not to be infected with HCV, based on consistently negativeresults with a first generation immunoassay test (Ortho HCV ELISA TestSystem; Ortho Diagnostics, Raritan, N.J.). However, subsequent testingusing a second generation HCV immunoassay (Ortho) and PCR with HCV5'-non-coding region primers demonstrated that the patient was infectedwith HCV.

III. ISOLATION OF HGV ASSOCIATED SEQUENCES.

As one approach toward identifying clones containing HGV sequences, aCDNA library was prepared from infected-HGV sera in the expressionvector lambda gt11 (Example 1). Polynucleotide sequences were thenselected for the expression of peptides which are immunoreactive withserum PNF 2161. First round screening was typically performed using thePNF 2161 serum (used to generate the phage library). It is also possibleto screen with other suspected N--(ABCDE) sera.

Recombinant proteins identified by this approach provide candidates forpeptides which can serve as substrates in diagnostic tests. Further, thenucleic acid coding sequences identified by this approach serve asuseful hybridization probes for the identification of additional HGVcoding sequences.

The sera described above were used to generate cDNA libraries in lambdagt11 (Example 1). In the method illustrated in Example 1, infected serumwas precipitated in 8% PEG without dilution, and the libraries weregenerated from the resulting pelleted virus. Sera from infected humansources were treated in the same fashion.

As an advantageous alternative to PEG precipitation, ultracentrifugationcan be used to pellet particulate agents from infected sera or otherbiological specimens. To isolate viral particles from which nucleicacids could be extracted, serum, ranging up to 2 ml, is diluted toapproximately 10 ml with PBS, spun at 3K for 10 minutes, and thesupernatant is centrifuged for a minimum of 2 hours at 40,000 rpm(approximately 110,000×g) in a Ti70.1 rotor (Beckman Instruments,Fullerton, Calif.) at 40° C. The supernatant is then aspirated and thepellet extracted by standard nucleic acid extraction techniques.

CDNA libraries were generated using random primers in reversetranscription reactions with RNA extracted from pelleted sera asstarting material. The resulting molecules were ligated to SequenceIndependent Single Primer Amplification (SISPA; Reyes, et al., 1991)linker primers and expanded in a non-selective manner, and then clonedinto a suitable vector, for example, lambda gt11, for expression andscreening of peptide antigens. Alternatively, the lambda gtlo vector mayalso be used.

Lambda gt11 is a particularly useful expression vector which contains aunique EcoRI insertion site 53 base pairs upstream of the translationtermination codon of the β-galactosidase gene. Thus, an insertedsequence is expressed as a β-galactosidase fusion protein which containsthe N-terminal portion of the β-galactosidase gene product, theheterologous peptide, and optionally the C-terminal region of theβ-galactosidase peptide (the C-terminal portion being expressed when theheterologous peptide coding sequence does not contain a translationtermination codon).

This vector also produces a temperature-sensitive repressor (cI857)which causes viral lysogeny at permissive temperatures, e.g., 32° C.,and leads to viral lysis at elevated temperatures, e.g., 42° C.Advantages of this vector include: (1) highly efficient recombinantclone generation, (2) ability to select lysogenized host cells on thebasis of host-cell growth at permissive, but not non-permissive,temperatures, and (3) production of recombinant fusion protein. Further,since phage containing a heterologous insert produces an inactiveβ-galactosidase enzyme, phage with inserts are typically identifiedusing a colorimetric substrate conversion reaction employingβ-galactosidase.

Example 1 describes the preparation of a CDNA library for the N-(ABCDE)hepatitis sera PNF 2161. The library was immunoscreened using PNF 2161(Example 3). A number of lambda gt11 clones were identified which wereimmunoreactive. Immunopositive clones were plaque-purified and theirimmunoreactivity retested. Also, the immunoreactivity of the clones withnormal human sera was also tested.

These clones were also examined for the "exogenous" nature of the clonedinsert sequence. This basic test establishes that the cloned fragmentdoes not represent a portion of human or other potentially contaminatingnucleic acids (e.g., E. coli, S. cerevisiea and mitochondrial). Theclone inserts were isolated by EcoRI digestion following polymerasechain reaction amplification. The inserts were purified thenradiolabelled and used as hybridization probes against membrane boundnormal human DNA, normal mystax DNA and bacterial DNA (control DNAs)(Example 4A).

Clone 470-20-1 (PNF2161 cDNA source) was one of the clones isolated byimmunoscreening with the PNF 2161 serum. The clone was not reactive withnormal human sera. The clone has a large open reading frame (203 basepairs; SEQ ID NO:3), in-frame with the β-galactosidase gene of thelambda gt11 vector. The clone is exogenous by genomic DNA hybridizationanalysis and genomic PCR analysis, using human, yeast and E. coligenomic DNAs (Example 4B).

The sequence was present in PNF2161 serum as determined by RT-PCR(Example 4C). RT-PCR of serially diluted PNF 2161 RNA suggested at leastabout 10⁵ copies of 470-20-1 specific sequence per ml. The sequence wasalso detected in sucrose density gradient fractions at densitiesconsistent with the sequence banding in association with a virus-likeparticle (Example 5).

Bacterial lysates of E. coli expressing a second clone, clone 470-expl,(SEQ ID NO:37) were also shown to be specifically immunoreactive withPNF 2161 serum at comparable levels to clone 470-20-1. The codingsequence of 470-exp1 was flanked by termination codons (based onsequence comparisons to SEQ ID NO:14, also see FIG. 1) and had aninternal methionine.

Further sequences contained in SEQ ID NO:14, adjacent to clone 470-20-1,were obtained by anchor polymerase chain reaction (Anchor PCR) usingprimers from clone 470-20-1 (Example 6). In this case a PNF 2161 2-cDNAsource library was used as template, where the cDNA/complementdouble-stranded DNA products were ligated to lambda arms, but themixture was not packaged. 470-20-1 specific primers were used inamplification reactions with SISPA-amplified PNF 2161 cDNA as a template(Example 4). The identity of the amplified DNA fragments were confirmedby (i) size and (ii) hybridization with a 470-20-1 specificoligonucleotide probe (SEQ ID NO:16). The 470-20-1 specific signal wasdetected in cDNA amplified by PCR from SISPA-amplified PNF 2161,demonstrating the presence of the 470-20-1 sequences in the sourcematerial.

The 470-20-1 specific primers were also used in amplification reactionswith the following RNA sources as substrate: normal mystax liver RNA,normal tamarin (Sanguins laboriatis) liver RNA, and MY131 liver RNA(Example 4). The results from these experiments demonstrate the 470-20-1sequences are present in the parent serum sample (PNF 2161) and in anRNA liver sample from an animal challenged with the PNF 2161 sample(MY131). Both normal control RNAs were negative for the presence of470-20-1 sequences.

Further, PNF 2161 serum and other cloning source or related sourcematerials were directly tested by PCR using primers from selected clonedsequences. Specific amplification products were detected byhybridization to a specific oligonucleotide probe 470-20-1-152F (SEQ IDNO:16). A specific signal was reproducibly detected in multiple extractsof PNF 2161, with the 470-20-1 specific primers.

The disease association between HGV and liver disease is furthersupported by the data presented in Example 4F. Sera from hepatitispatients and from blood donors with abnormal liver function wereassessed for the presence of HGV by RT-PCR screening, using HGV specificprimers. HGV specific sequence were detected in 6/152 of these serasamples. No HGV positives were detected among the control samples(n=11).

The results presented above indicate the isolation of a viral agentassociated with N-(ABCDE) viral infection of liver (i.e., hepatitis)and/or infection, and resulting disease, of other tissue and cell types.

IV. FURTHER CHARACTERIZATION OF HGV RECOMBINANT ANTIGENS.

A. Screening Recombinant Libraries.

Further candidate HGV antigens can be obtained from the libraries of thepresent invention using the screening methods described above. The CDNAlibrary described above has been deposited with the American TypeCulture Collection, 12301 Parklawn Dr., Rockville, Md., 20852, and hasbeen assigned the following designation: PNF 2161 cDNA source, ATCC75268. The deposit was accepted by the International DepositoryAuthority on Jul. 16, 1992.

A second PNF 2161 DNA library has been generated essentially asdescribed for the first PNF 2161 cDNA library, except that second PNF2161 cDNA source library was ligated to lambda gt11 arms but was notpackaged. This non-packaged library was used to obtain the extensionclones described below. A packaged version of this second library (PNF2161 2-cDNA source library) has been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., 20852, and hasbeen assigned the following designation: PNF 2161 2-cDNA source, ATCC75837. c4

In addition to the recombinant libraries generated above, otherrecombinant libraries from N-(ABCDE) hepatitis sera can likewise begenerated and screened as described herein.

B. Epitope Mapping, Cross Hybridization and Isolation of GenomicSequences.

Antigen encoding DNA fragments can be identified by (i) immunoscreening,as described above, or (ii) computer analysis of coding sequences (e.g.,SEQ ID NO:14) using an algorithm (such as, "ANTIGEN," Intelligenetics,Mountain View, Calif.) to identify potential antigenic regions. Anantigen-encoding DNA fragment can be subcloned. The subcloned insert canthen be fragmented by partial DNase I digestion to generate randomfragments or by specific restriction endonuclease digestion to producespecific subfragments. The resulting DNA fragments can be inserted intothe lambda gt11 vector and subjected to immunoscreening in order toprovide an epitope map of the cloned insert.

In addition, the DNA fragments can be employed as probes inhybridization experiments to identify overlapping HGV sequences, andthese in turn can be further used as probes to identify a set ofcontiguous clones. The generation of sets of contiguous clones allowsthe elucidation of the sequence of the HGV's genome.

Any of the above-described clone sequences (e.g., derived from SEQ IDNO:14 or clone 470-20-1) can be used to probe the cDNA and DNAlibraries, generated in a vector such as lambda gt10 or "LAMBDA ZAP II"(Stratagene, San Diego, Calif.). Specific subfragments of known sequencemay be isolated by polymerase chain reaction or after restrictionendonuclease cleavage of vectors carrying such sequences. The resultingDNA fragments can be used as radiolabelled probes against any selectedlibrary. In particular, the 5' and 3' terminal sequences of the cloneinserts are useful as probes to identify additional clones.

Further, the sequences provided by the 5' end of cloned inserts areuseful as sequence specific primers in first-strand CDNA or DNAsynthesis reactions (Maniatis et al.; Scharf et al.). For example,specifically primed PNF 2161 cDNA and DNA libraries can be prepared byusing specific primers derived from SEQ ID NO:14 on PNF 2161 nucleicacids as a template. The second-strand of the new cDNA is synthesizedusing RNase H and DNA polymerase I. The above procedures identify orproduce DNA/cDNA molecules corresponding to nucleic acid regions thatare 5' adjacent to the known clone insert sequences. These newlyisolated sequences can in turn be used to identify further flankingsequences, and so on, to identify the sequences composing the entiregenome for HGV. As described above, after new HGV sequences areisolated, the polynucleotides can be cloned and immunoscreened toidentify specific sequences encoding HGV antigens.

Extension clone sequences (SEQ ID NO:14), containing further sequencesof interest, have been obtained for clone PNF 470-20-1 (SEQ ID NO:3)using the "Anchor PCR" method described in Example 6. Briefly, thestrategy consists of ligating PNF 2161 SISPA cDNA to lambda gt11 armsand amplifying the ligation reaction with a gt11-specific primer and oneof two 470-20-1 specific primers.

The amplification products are electrophoretically separated,transferred to filters and the DNA bound to the filters is probed with a470-20-1 specific probe. Bands corresponding to hybridization positiveband signals were gel purified, cloned and sequenced.

C. Preparation of Antigenic Polypeptides and Antibodies.

The recombinant peptides of the present invention can be purified bystandard protein purification procedures which may include differentialprecipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis and affinitychromatography.

In one embodiment of the present invention, the polynucleotide sequencesof the antigens of the present invention have been cloned in the plasmidp-GEX (Example 7A) or various derivatives thereof (pGEX-GLI). Theplasmid pGEX (Smith, et al., 1988) and its derivatives express thepolypeptide sequences of a cloned insert fused in-frame to the proteinglutathione-S-transferase (sj26). In one vector construction, plasmidpGEX-hisB, an amino acid sequence of 6 histidines is introduced at thecarboxy terminus of the fusion protein.

The various recombinant PGEX plasmids can be transformed intoappropriate strains of E. coli and fusion protein production can beinduced by the addition of IPTG (isopropyl-thio galactopyranoside) asdescribed in Example 7A. Solubilized recombinant fusion protein can thenbe purified from cell lysates of the induced cultures using glutathioneagarose affinity chromatography (Example 7A).

Insoluble fusion protein expressed by the plasmid pGEX-hisB can bepurified by means of immobilized metal ion affinity chromatography(Porath) in buffers containing 6M Urea or 6M guanidinium isothiocyanate,both of which are useful for the solubilization of proteins.Alternatively insoluble proteins expressed in pGEX-GLI or derivativesthereof can be purified using combinations of centrifugation to removesoluble proteins followed by solubilization of insoluble proteins andstandard chromatographic methodologies, such as ion exchange or sizeexclusion chromatography, and other such methods are known in the art.

In the case of β-galactosidase fusion proteins (such as those producedby lambda gt11 clones) the fused protein can be isolated readily byaffinity chromatography, by passing cell lysis material over a solidsupport having surface-bound anti-β-galactosidase antibody. For example,purification of a β-galactosidase/fusion protein, derived from 470-20-1coding sequences, by affinity chromatography is described in Example 7B.

Also included in the invention is an expression vector, such as thelambda gt11 or pGEX vectors described above, containing HGV codingsequences and expression control elements which allow expression of thecoding regions in a suitable host. The control elements generallyinclude a promoter, translation initiation codon, and translation andtranscription termination sequences, and an insertion site forintroducing the insert into the vector.

The DNA encoding the desired antigenic polypeptide can be cloned intoany number of commercially available vectors to generate expression ofthe polypeptide in the appropriate host system. These systems include,but are not limited to, the following: baculovirus expression (Reilly,et al.; Beames, et al.; Pharmingen; Clontech, Palo Alto, Calif.),vaccinia expression (Earl, 1991; Moss, et al.), expression in bacteria(Ausubel, et al.; Clontech), expression in yeast (Gellissen, 1992;Romanos, 1992; Goeddel; Guthrie and Fink), expression in mammalian cells(Clontech; Gibco-BRL, Ground Island, N.Y.), e.g., Chinese hamster ovary(CHO) cell lines (Haynes, 1983, Lau, 1984, Kaufman, 1990). Theserecombinant polypeptide antigens can be expressed directly or as fusionproteins. A number of features can be engineered into the expressionvectors, such as leader sequences which promote the secretion of theexpressed sequences into culture medium.

Expression of large HGV polypeptides using several of these systems isdescribed in Example 16.

Expression in yeast systems has the advantage of commercial production.Recombinant protein production by vaccinia and CHO cell line have theadvantage of being mammalian expression systems. Further, vaccinia virusexpression has several advantages including the following: (i) its widehost range; (ii) faithful post-transcriptional modification, processing,folding, transport, secretion, and assembly of recombinant proteins;(iii) high level expression of relatively soluble recombinant proteins;and (iv) a large capacity to accommodate foreign DNA.

The recombinant expressed polypeptide produced HGV polypeptide antigensare typically isolated from lysed cells or culture media. Purificationcan be carried out by methods known in the art including saltfractionation, ion exchange chromatography, and affinity chromatography.Immunoaffinity chromatography can be employed using antibodies generatedbased on the HGV antigens identified by the methods of the presentinvention.

HGV polypeptide antigens may also be isolated from HGV particles (seebelow).

Continuous antigenic determinants of polypeptides are generallyrelatively small, typically 6 to 10 amino acids in length. Smallerfragments have been identified as antigenic regions, for example, inconformational epitopes. HGV polypeptide antigens are identified asdescribed above. The resulting DNA coding regions of either strand canbe expressed recombinantly either as fusion proteins or isolatedpolypeptides. In addition, amino acid sequences can be convenientlychemically synthesized using commercially available synthesizer (AppliedBiosystems, Foster City, Calif.) or "PIN" technology (AppliedBiosytems).

In another embodiment, the present invention includes mosaic proteinsthat are composed of multiple epitopes. An HGV mosaic polypeptidetypically contains at least two epitopes of HGV, where the polypeptidesubstantially lacks amino acids normally intervening between theepitopes in the native HGV coding sequence. Synthetic genes (Crea;Yoshio et al.; Eaton et al.) encoding multiple, tandem epitopes can beconstructed that will produce mosaic proteins using standard recombinantDNA technology using polypeptide expression vector/host system describedabove.

Further, multiple antigen peptides can be synthesized chemically bymethods described previously (Tam, J. P., 1988; Briand et al.). Forexample, a small immunologically inert core matrix of lysine residueswith α- and ε- amino groups can be used to anchor multiple copies of thesame or different synthetic peptides (typically 6-15 residues long)representing epitopes of interest. Mosaic proteins or multiple antigenpeptide antigens give higher sensitivity and specificity in immunoassaysdue to the signal amplification resulting from distribution of multipleepitopes.

Antigens obtained by any of these methods can be used for antibodygeneration, diagnostic tests and vaccine development.

In another aspect, the invention includes specific antibodies directedagainst the polypeptide antigens of the present invention. Antigensobtained by any of these methods may be directly used for the generationof antibodies or they may be coupled to appropriate carrier molecules.Many such carriers are known in the art and are commercially available(e.g., Pierce, Rockford Ill.). Typically, to prepare antibodies, a hostanimal, such as a rabbit, is immunized with the purified antigen orfused protein antigen. Hybrid, or fused, proteins may be generated usinga variety of coding sequence derived from other proteins, such asglutathione-S-transferase or β-galactosidase. The host serum or plasmais collected following an appropriate time interval, and this serum istested for antibodies specific against the antigen. Example 8 describesthe production of rabbit serum antibodies which are specific against the470-20-1 antigen in the Sj26/470-20-1 hybrid protein. These techniquesare equally applicable to all immunogenic sequences derived from HGV,including, but not limited to, those derived from the coding sequencepresented as SEQ ID NO:14.

The gamma globulin fraction or the IgG antibodies of immunized animalscan be obtained, for example, by use of saturated ammonium sulfateprecipitation or DEAE Sephadex chromatography, affinity chromatography,or other techniques known to those skilled in the art for producingpolyclonal antibodies.

Alternatively, purified antigen or fused antigen protein may be used forproducing monoclonal antibodies. Here the spleen or lymphocytes from animmunized animal are removed and immortalized or used to preparehybridomas by methods known to those skilled in the art. To produce ahuman-derived hybridoma, a human lymphocyte donor is selected. A donorknown to be infected with a HGV may serve as a suitable lymphocytedonor. Lymphocytes can be isolated from a peripheral blood sample.Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes ora suitable fusion partner can be used to produce human-derivedhybridomas. Primary in vitro sensitization with viral specificpolypeptides can also be used in the generation of human monoclonalantibodies.

Antibodies secreted by the immortalized cells are screened to determinethe clones that secrete anti-bodies of the desired specificity, forexample, by using the ELISA or Western blot method (Example 10; Ausubelet al.).

Using HGV-positive serum or plasma, or the antibodies of the presentinvention, other antigenic peptides and epitopes can be isolated. Forexample, a number of different techniques have been developed for thesimultaneous synthesis of many peptides (Geysen, et al.; Houghten; Frankand Doring; Hudson). The method developed by Geysen, et al., isespecially useful because of the relative simplicity with which largenumbers of different peptide sequences can be generated and tested forantigenicity. In the Geysen method (also referred to as MULTI-PINpeptide synthesis), the peptides are synthesized on polyacrylamide acidgrafted polyethylene rods attached to a micro-titer plate. The MULTI-PINstrategy allows large numbers of syntheses (96 peptides per plate) to beimmunologically screened using the polyclonal or monoclonal antibodiesof the present invention and commercially available reagents andinstrumentation. Immunoreactive peptides are identified andcharacterized.

It has been reported that up to 6,000 oligopeptides can be synthesizedin a two week period, thus making it practical (by synthesizing all ofthe possible overlapping amino acid sequences of a particular antigen)to screen viral antigen sequences for epitopes to the resolution of asingle amino acid (Geysen, et al.).

An alternative method of scanning for immunodominate peptides is tosynthesize longer peptides (e.g., 10 to 30 amino acids) corresponding toHGV coding sequences using conventional automated peptide synthesis(Carter, et al., 1994; Obeid, et al., 1994; Commandaeur, et al., 1994).This method has the advantage that the longer peptides can fold intoshapes that mimic conformational epitopes.

Also, HGV antibodies, in particular, monoclonals, can be used toidentify random polypeptides that mimic their virus-encoded targetpolypeptides (Scott and Smith, 1990; Smith, 1991). For example, randompeptide libraries displayed on phage (RPL) (Scott and Smith, 1990) canbe used as a source of peptide ligands for antibody generation or forvaccine development. RPL approach allows the expression ofpeptide-ligand containing fusion proteins on the phage surface andenrichment of these ligand encoding phages by affinity selection usingantibodies (Smith, J.P., 1991; Christian, et al.; Scott, et al., 1992;Folgori, et al.). These random peptide epitopes detected by specificantibodies mimic the natural antigenic epitopes (mimotopes) duringepitope presentation. HGV antigenic mimics (mimotopes) can be isolatedfrom RPL. Hexa- to decapeptide phagotope (mimotope displayed on phage)expressing RPL can be made by published methods (Scott and Smith; Smith,J.P, 1991; Christian, et al.; Scott, et al.; DeGraaf, et al.; Folgori,et al.) and screened by HGV-associated human sera or the antibodies ofthe present invention.

One example of the use of RPL for isolation of 470-20-1 mimotopes is asfollows. The random decapeptide-pIII fusion phage display library isconstructed according to the methods described previously (DeGraaf, etal., 1993). Briefly, a chemically synthesized single-stranded degenerateinsert is annealed to shorter oligonucleotides which generate SfiIrestriction overhangs. Annealed DNA is ligated into SfiI-cut fUSE-5vector DNA.

E. coli MC1061 is transformed with the ligated DNA. The library isamplified through approximately ten population doublings in LB mediumwith 20 mg/ml tetracycline. This library is affinity selected using oneor more of 470-20-1 immunoreactive sera (or antibodies of the presentinvention). Polystyrene beads (Precision Plastic Ball Company, Chicago.Ill.) are coated with ammonium sulfate fractionated positive serum(e.g., PNF 2161) in 50 mM NaHCO3, pH 9.6 overnight at 4° C. Antibodycoated beads are thoroughly washed with PBS and blocked with BSA.

These serum coated, blocked beads are pre-incubated with an excess ofM13K07-UV killed phage for 4 hours at 40° C. Library phage are thenadded to the above pre-incubation mixture and incubated for 12 hours at4° C. Unbound phage are removed and the beads are washed extensivelywith TTB (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% "TWEEN 20" (v/v), 1mg/ml BSA) buffer. Bound phage are eluted with elution buffer (0.1M HCladjusted to pH 2.2 with 2M Tris-HCl, pH 9.0). Eluted, enriched phage arescreened with a second positive serum (e.g., Mys 136 sera) by plaqueimmunoscreening.

Further screening of the selected phagotopes can be carried out usinglarge panels of positive and negative sera or specific HGV monoclonalantibodies. Selected phagotopes can be used directly in ELISA assay orantibody generation. Alternatively, the sequences of the phagotopeencoding nucleotides can be determined and expressed in conventionalvector/host system and used as antigen.

Mimic polypeptides identified as described above can in turn can serveas antigens in detection assays or can be used for the generation ofantigen-specific antibodies.

D. Elisa and Protein Blot Screening.

When HGV antigens are identified, typically through plaqueimmunoscreening as described above, the antigens can be expressed andpurified. The antigens can then be screened rapidly against a largenumber of suspected HGV hepatitis sera using alternative immunoassays,such as, ELISAs or Protein Blot Assays (Western blots) employing theisolated antigen peptide. The antigen polypeptides fusion can beisolated as described above, usually by affinity chromatography to thefusion partner such as β-galactosidase or glutathione-S-transferase.Alternatively, the antigen itself can be purified using antibodiesgenerated against it (see below).

A general ELISA assay format is presented in Example 10. Harlow, et al.,describe a number of useful techniques for immunoassays andantibody/antigen screening.

The purified antigen polypeptide or fusion polypeptide containing theantigen of interest, is attached to a solid support, for example, amultiwell polystyrene plate. Sera to be tested are diluted and added tothe wells. After a period of time sufficient for the binding ofantibodies to the bound antigens, the sera are washed out of the wells.A labelled reporter antibody is added to each well along with anappropriate substrate: wells containing antibodies bound to the purifiedantigen polypeptide or fusion polypeptide containing the antigen aredetected by a positive signal.

A typical format for protein blot analysis using the polypeptideantigens of the present invention is presented in Example 10. Generalprotein blotting methods are described by Ausubel, et al. In Example 10,the 470-20-1/sj26 fusion protein was used to screen a number of serasamples. The results presented in Example 10 demonstrate that severaldifferent source N-(ABCDE) hepatitis sera are immunoreactive with thepolypeptide antigen.

The results presented above demonstrate that the polypeptide antigens ofthe present invention can, by these methods, be rapidly screened againstpanels of suspected HGV infected serum samples for the detection of HGV.

E. Cell Culture Systems, Animal Models and Isolation of HGV.

HGV infectivity studies have been carried out in chimpanzees, cynomolgusmonkey and-four mystax subjects (Example 4H). These studies have yieldedfurther information about HGV infectivity in these animal models. TheHGV described in the present specification have the advantage of beingcapable of infecting tamarins, cynomologous monkeys and chimpanzees.

Alternatively, primary hepatocytes obtained from infected animals(chimpanzees, baboons, monkeys, or humans) can be cultured in vitro. Aserum-free medium, supplemented with growth factors and hormones, hasbeen described which permits the long-term maintenance of differentiatedprimate hepatocytes (Lanford, et al.; Jacob, et al., 1989, 1990, 1991).In addition to primary hepatocyte cultures, immortalized cultures ofinfected cells may also be generated. For example, primary livercultures may be fused to a variety of cells (like HepG2) to providestable immortalized cell lines. Primary hepatocyte cell cultures mayalso be immortalized by introduction of oncogenes or genes causing atransformed phenotype. Such oncogenes or genes can be derived from anumber of sources known in the art including SV40, human cellularoncogenes and Epstein Barr Virus.

Further, the un-infected hepatocytes (e.g., primary or continuoushepatoma cell lines) may be infected by exposing the cells in culture tothe HGV either as partially purified particle preparations (prepared,for example, from infected sera by differential centrifugation and/ormolecular sieving) or in infectious sera. These infected cells can thenbe propagated and the virus passaged by methods known in the art. Inaddition, other cell types, such as lymphoid cell lines, may be usefulfor the propagation of HGV.

Protein similarity studies of HGV have detected amino acid regionssimilar to other viruses in the family Flaviviridae. It is known thatmembers of this family of viruses can be propagated in a variety oftissue culture systems (ATCC-Viruses catalogue, 1990). By analogy it islikely that HGV can be propagated in one or more of the following tissueculture systems: Hela cells, primary hamster kidney cells, monkey kidneycells, vero cells, LLC-MK2 (rhesus monkey kidney cells), KB cells(humanoral epidermoid carcinoma cells), duck embryo cells, primary sheepleptomeningeal cells, primary sheep choroid plexus cells, pig kidneycells, bovine embryonic kidney cells, bovine turbinate cells, chickembryo cells, primary rabbit kidney cells, BHD-21 cells, or PK-13 cells.

In addition to expression of HGV, regions of HGV polynucleotidesequences, cDNA or in vitro transcribed RNA can be introduced byrecombinant means into tissue culture cells. Such recombinantmanipulations allow the individual expression of individual componentsof the HGV.

RNA samples can be prepared from infected tissue or, in particular, frominfected cell cultures. The RNA samples can be fractionated on gels andtransferred to membranes for hybridization analysis using probes derivedfrom the cloned HGV sequences.

HGV particles may be isolated from infected sera, infected tissue, theabove-described cell culture media, or the cultured infected cells bymethods known in the art. Such methods include techniques based on sizefractionation (i.e., ultrafiltration, precipitation, sedimentation),using anionic and/or cationic exchange materials, separation on thebasis of density, hydrophilic properties, and affinity chromatography.During the isolation procedure the HGV can be identified (i) using theanti-HGV hepatitis associated agent antibodies of the present invention,(ii) by using hybridization probes based on identified HGV nucleic acidsequences (e.g., Example 5) or (iii) by RT-PCR.

Antibodies directed against HGV can be used in purification of HGVparticles through immunoaffinity chromatography (Harlow, et al.;Pierce). Antibodies directed against HGV polypeptides or fusionpolypeptides (such as 470-20-1) are fixed to solid supports in such amanner that the antibodies maintain their immunoselectivity. Toaccomplish such attachment of antibodies to solid support bifunctionalcoupling agents (Pierce; Pharmacia, Piscataway, N.J.) containing spacergroups are frequently used to retain accessibility of the antigenbinding site of the antibody.

HGV particles can be further characterized by standard proceduresincluding, but not limited to, immunofluorescence microscopy, electronmicroscopy, Western blot analysis of proteins composing the particles,infection studies in animal and/or cell systems utilizing the partiallypurified particles, and sedimentation characteristics. The resultspresented in Example 5 suggest that the viral particle of the presentinvention is more similar to an enveloped viral particle than to anon-enveloped viral particle.

HGV particles can be disrupted to obtain HGV genomes. Disruption of theparticles can be achieved by, for example, treatment with detergents inthe presence of chelating agents. The genomic nucleic acid can then befurther characterized. Characterization may include analysis of DNaseand RNase sensitivity. The strandedness (Example 4I) and conformation(e.g., circular) of the genome can be determined by techniques known inthe art, including visualization by electron microscopy andsedimentation characteristics.

The isolated genomes also make it possible to sequence the entire genomewhether it is segmented or not, and whether it is an RNA or DNA genome(using, for example RT-PCR, chromosome walking techniques, or PCR whichutilizes primers from adjacent cloned sequences). Determination of theentire sequence of HGV allows genomic organization studies and thecomparison of the HGV sequences to the coding and regulatory sequencesof known viral agents.

F. Screening for Agents having Anti-HGV Hepatitis Activity.

The use of cell culture and animal model systems for propagation of HGVprovides the ability to screen for anti-hepatitis agents which inhibitthe production of infectious HGV: in particular, drugs that inhibit thereplication of HGV. Cell culture and animal models allow the evaluationof the effect of such anti-hepatitis drugs on normal cellular functionsand viability. Potential anti-viral agents (including natural productsor synthetic compounds; for example, small molecules, complex mixturessuch as fungal extracts, and anti-sense oligonucleotides) are typicallyscreened for anti-viral activity over a range of concentrations. Theeffect on HGV replication and/or antigen production is then evaluated,typically by monitering viral macromolecular synthesis or accumulationof macromolecules (e.g., DNA, RNA or protein). This evaluation is oftenmade relative to the effect of the anti-viral agent on normal cellularfunction (DNA replication, RNA transcription, general proteintranslation, etc.).

The detection of the HGV can be accomplished by many methods includingthose described in the present specification. For example, antibodiescan be generated against the antigens of the present invention and theseantibodies used in antibody-based assays (Harlow, et al.) to identifyand quantitate HGV antigens in cell culture. HGV antigens can bequantitated in culture using competition assays: polypeptides encoded bythe cloned HGV sequences can be used in such assays. Typically, arecombinantly produced HGV antigenic polypeptide is produced and used togenerate a monoclonal or polyclonal antibody. The recombinant HGVpolypeptide is labelled using a reporter molecule. The inhibition ofbinding of this labelled polypeptide to its cognate antibody is thenevaluated in the presence of samples (e.g., cell culture media or sera)that contain HGV antigens. The level of HGV antigens in the sample isdetermined by comparison of levels of inhibition to a standard curvegenerated using unlabelled recombinant proteins at known concentrations.

The HGV sequences of the present invention are particularly useful forthe generation of polynucleotide probes/primers that may be used toquantitate the amount of HGV nucleic acid sequences produced in a cellculture system. Such quantification can be accomplished in a number ofways. For example, probes labelled with reporter molecules can be usedin standard dot-blot hybridizations or competition assays of labelledprobes with infected cell nucleic acids. Further, there are a number ofmethods using the polymerase chain reaction to quantitate target nucleicacid levels in a sample (Osikowicz, et al.).

Protective antibodies can also be identified using the cell culture andanimal model systems described above. For example, polyclonal ormonoclonal antibodies are generated against the antigens of the presentinvention. These antibodies are then used to pre-treat an infectiousHGV-containing inoculum (e.g., serum) before infection of cell culturesor animals. The ability of a single antibody or mixtures of antibodiesto protect the cell culture or animal from infection is evaluated. Forexample, in cell culture and animals the absence of viral antigen and/ornucleic acid production serves as a screen. Further in animals, theabsence of HGV hepatitis disease symptoms, e.g., elevated ALT values, isalso indicative of the presence of protective antibodies.

Alternatively, convalescent sera can be screened for the presence ofprotective antibodies and then these sera used to identify HGV hepatitisassociated agent antigens that bind with the antibodies. The identifiedHGV antigen is then recombinantly or synthetically produced. The abilityof the antigen to generate protective antibodies is tested as above.

After initial screening, the antigen or antigens identified as capableof generating protective antibodies, either singly or in combination,can be used as a vaccine to inoculate test animals. The animals are thenchallenged with infectious HGV. Protection from infection indicates theability of the animals to generate antibodies that protect them frominfection. Further, use of the animal models allows identification ofantigens that activate cellular immunity.

In animal model studies, a protective immune response in response tochallenge by a viral preparation (e.g., infected serum) (i) protects theanimal from infection or (ii) prevents manifestation of disease.

G. Vaccines and the Generation of Protective Immunity.

Vaccines can be prepared from one or more of the immunogenicpolypeptides identified by the method of the present invention. Genomicorganization similarities between the isolated sequences from HGV andother known viral proteins may provide information concerning thepolypeptides that are likely to be candidates for effective vaccines. Inaddition, a number of computer programs can be used for to identifylikely regions of isolated sequences that encode protein antigenicdeterminant regions (for example, Hopp, et al.; "ANTIGEN,"Intelligenetics, Mountain View Calif.).

Vaccines containing immunogenic polypeptides as active ingredients aretypically prepared as injectables either as solutions or suspensions.Further, the immunogenic polypeptides may be prepared in a solid orlyophilized state that is suitable for resuspension, prior to injection,in an aqueous form. The immunogenic polypeptides may also be emulsifiedor encapsulated in liposomes. The polypeptides are frequently mixed withpharmaceutically acceptable excipients that are compatible with thepolypeptides. Such excipients include, but are not limited to, thefollowing and combinations of the following: saline, water, sugars (suchas dextrose and sorbitol), glycerol, alcohols (such as ethanol EtOH!),and others known in the art. Further, vaccine preparations may containminor amounts of other auxiliary substances such as wetting agents,emulsifying agents (e.g., detergents), and pH buffering agents. Inaddition, a number of adjuvants are available which may enhance theeffectiveness of vaccine preparations. Examples of such adjuvantsinclude, but are not limited to, the following: the group of relatedcompounds including N-acetyl-muranyl-L-threonyl-D-isoglutamine andN-acetyl-nor-muranyl-L-alanyl-D-isoglutamine, and aluminum hydroxide.

The immunogenic polypeptides used in the vaccines of the presentinvention may be recombinant, synthetic or isolated from, for example,attenuated HGV particles. The polypeptides are commonly formulated intovaccines in neutral or salt forms. Pharmaceutically acceptable organicand inorganic salts are well known in the art.

HGV hepatitis associated agent vaccines are parenterally administered,typically by subcutaneous or intramuscular injection. Other possibleformulations include oral and suppository formulations. Oralformulations commonly employ excipients (e.g., pharmaceutical gradesugars, saccharine, cellulose, and the like) and usually contain within10-98% immunogenic polypeptide. Oral compositions take the form ofpills, capsules, tablets, solutions, suspensions, powders, etc., and maybe formulated to allow sustained or long-term release. Suppositoryformulations use traditional binders and carriers and typically containbetween 0.1% and 10% of the immunogenic polypeptide.

In view of the above information, multivalent vaccines against HGVhepatitis associated agents can be generated which are composed of oneor more structural or non-structural viral-agent polypeptide(s). Thesevaccines can contain, for example, recombinant expressed HGVpolypeptides, polypeptides isolated from HGV virions, syntheticpolypeptides or assembled epitopes in the form of mosaic polypeptides.In addition, it may be possible to prepare vaccines, which conferprotection against HGV hepatitis infection through the use ofinactivated HGV. Such inactivation might be achieved by preparation ofviral lysates followed by treatment of the lysates with appropriateorganic solvents, detergents or formalin.

Vaccines may also be prepared from attenuated HGV strains. Suchattenuated HGV may be obtained utilizing the above described cellculture and/or animal model systems. Typically, attenuated strains areisolated after multiple passages in vitro or in vivo. Detection ofattenuated strains is accomplished by methods known in the art. Onemethod for detecting attenuated HGV is the use of antibody probesagainst HGV antigens, sequence-specific hybridization probes, oramplification with sequence-specific primers for infected animals orassay of HGV-infected in vitro cultures.

Alternatively, or in addition to the above methods, attenuated HGVstrains may be constructed based on the genomic information that can beobtained from the information presented in the present specification.Typically, a region of the infectious agent genome that encodes, forexample, a polypeptide that is related to viral pathogenesis can bedeleted. The deletion should not interfere with viral replication.Further, the recombinant attenuated HGV construct allows the expressionof an epitope or epitopes that are capable of giving rise to protectiveimmune responses against the HGV. The desired immune response mayinclude both humeral and cellular immunity. The genome of the attenuatedHGV is then used to transform cells and the cells grown under conditionsthat allow viral replication. Such attenuated strains are useful notonly as vaccines, but also as production sources of viral antigensand/or HGV particles.

Hybrid particle immunogens that contain HGV epitopes can also begenerated. The immunogenicity of HGV epitopes may be enhanced byexpressing the epitope in eucaryotic systems (e.g., mammalian or yeastsystems) where the epitope is fused or assembled with known particleforming proteins. One such protein is the hepatitis B surface antigen.Recombinant constructs where the HGV epitope is directly linked tocoding sequence for the particle forming protein will produce hybridproteins that are immunogenic with respect to the HGV epitope and theparticle forming protein. Alternatively, selected portions of theparticle-forming protein coding sequence, which are not involved inparticle formation, may be replaced with coding sequences correspondingto HGV epitopes. For example, regions of specific immunoreactivity tothe particle-forming protein can be replaced by HGV epitope sequences.

The hepatitis B surface antigen has been shown to be expressed andassembled into particles in the yeast Saccharomyces cerevisiea and inmammalian cells (Valenzuela, et al., 1982 and 1984; Michelle, et al.).These particles have been shown to have enhanced immunoreactivity.Formation of these particles using hybrid proteins, i.e., recombinantconstructs with heterologous viral sequences, has been previouslydisclosed (EPO 175,261, published Mar. 26, 1986). Such hybrid particlescontaining HGV epitopes may also be useful in vaccine applications.

The vaccines of the present invention are administered in dosagescompatible with the method of formulation, and in such amounts that willbe pharmacologically effective for prophylactic or therapeutictreatments. The quantity of immunogen administered depends on thesubject being treated, the capacity of the treatment subject's immunesystem for generation of protective immune response, and the desiredlevel of protection.

HGV vaccines of the present invention can be administered in single ormultiple doses. Dosage regimens are also determined relative to thetreatment subject's needs and tolerances. In addition to the HGVimmunogenic polypeptides, vaccine formulations may be administered inconjunction with other immunoregulatory agents.

In an additional approach to HGV vaccination, DNA constructs encodingHGV proteins under appropriate regulatory control are introduceddirectly into mammalian tissue, in vivo. Introduction of such constructsproduces "genetic immunization". Similar DNA constructs have been shownto be taken up by cells and the encoded proteins expressed (Wolf, etal.; Ascadi, et al.). Injected DNA does not appear to integrate intohost cells chromatin or replicate. This expression gives rise tosubstantial humoral and cellular immune responses, including protectionfrom in vivo viral challenge in animal systems (Wang, et al., 1993;Ulmer, et al.). In one embodiment, the DNA construct is injected intoskeletal muscle following pre-treatment with local anesthetics, such as,bupivicaine hydrochloride with methylparaben in isotonic saline, tofacilitate cellular DNA uptake. The injected DNA constructs are taken upby muscle cells and the encoded proteins expressed.

Compared to vaccination with soluble viral subunit proteins, geneticimmunization has the advantage of authentic in vivo expression of theviral proteins. These viral proteins are expressed in association withhost cell histocompatibility antigens, and other proteins, as wouldoccur with natural viral infection. This type of immunization is capableof inducing both humoral and cellular immune responses, in contrast tomany soluble subunit protein vaccines. Accordingly, this type ofimmunization retains many of the beneficial features of live attenuatedvaccines, without the use of infectious agents for vaccination andattendant safety concerns.

Direct injection of plasmid or other DNA constructs encoding the desiredvaccine antigens into in vivo tissues is one delivery means. Other meansof delivery of the DNA constructs can be employed as well. These includea variety of lipid-based approaches in which the DNA is packaged usingliposomes, cationic lipid reagents or cytofectins (such as, lipofectin).These approaches facilitate in vivo uptake and expression, as summarizedby Felgner and Rhodes (1991). Various modifications to these basicapproaches include the following: incorporation of peptides, or othermoieties, to facilitate (i) targeting to particular cells, (ii) theintracellular disposition of the DNA construct following uptake, or(iii) to facilitate expression. Alternatively, the sequences encodingthe desired vaccine antigens may be inserted into a suitable retroviralvector. The resulting recombinant retroviral vector inoculated into thesubject for in vivo expression of the vaccine antigen. The antigen theninduces the immune responses. As noted above, this approach has beenshown to induce both humoral and cellular immunity to viral antigens(Irwin, et al.).

Further, the HGV vaccines of the present invention may be administeredin combination with other vaccine agents, for example, with otherhepatitis vaccines.

H. Synthetic Peptides.

Using the coding sequences of HGV polypeptide, synthetic peptides can begenerated which correspond to these polypeptides. Synthetic peptides canbe commercially synthesized or prepared using standard methods andapparatus in the art (Applied Biosystems, Foster City Calif.).

Alternatively, oligonucleotide sequences encoding peptides can be eithersynthesized directly by standard methods of oligonucleotide synthesis,or, in the case of large coding sequences, synthesized by a series ofcloning steps involving a tandem array of multiple oligonucleotidefragments corresponding to the coding sequence (Crea; Yoshio et al.;Eaton et al.). Oligonucleotide coding sequences can be expressed bystandard recombinant procedures (Maniatis et al.; Ausubel et al.).

V. CHARACTERIZATION OF THE VIRAL GENOME.

As shown in Example 4, the HGV genome appears to be an RNA molecule andhas the closest sequence similarity to viral sequences that arecategorized in the Flaviviridae family of viruses. This family includesthe Flaviviruses, Pestiviruses and an unclassified Genus made up of onemember, Hepatitis C virus. The HGV virus does not have significantglobal (i.e., over the length of the virus) sequence identity with otherrecognized members of the Flaviviridae--with the exception of theprotein motifs discussed below.

In general members of the Flaviviridae are enveloped viruses that havedensities in sucrose gradients between 1.1 and 1.23 g/ml and aresensitive to heat, organic solvents and detergents. As shown in Example5, HGV has density characteristics similar to an enveloped Flaviviridaevirus (HCV). The integrity of the HGV virion also appears to besensitive to organic solvents (Example 5).

Flaviviridae virions contain a single molecule of linear single-stranded(ss) RNA which also serves as the only mRNA that codes for the viralproteins. The ssRNA molecule is typically between the size of 9 and 12kilobases long.

Viral proteins are derived from one polyprotein precursor that issubsequently processed to the mature viral proteins. Most members of theFlaviviridae do not contain poly(A) tails at their 3' ends. Virions areabout 15-20% lipid by weight.

Members in the Flaviviridae family have a core protein and two or threemembrane-associated proteins. The analogous structural proteins ofmembers in the three genera Flavivirus family show little similarity toone another at the sequence level. The nonstructural proteins containconserved motifs for RNA dependent RNA polymerase (RDRP), helicase, anda serine protease. These short blocks of conserved amino acids or motifscan be detected using computer algorithms known in the art such as"MACAW" (Schuler, et al.). These motifs are presumably related toconstraints imposed by substrates processed by these proteins (Kooninand Dolja). The order of these motifs is conserved in all members of theFlaviviridae family. The genome of HGV contains protein motifs found inmembers of the Flaviviridae family, for example, (i) the helicase gene,(ii) the serine-like protease domain, and (iii) the RNA dependent RNApolymerase (RDRP) of (see FIG. 5, "GDD" sequence);

Sequence information is disclosed herein on several differentstrains/isolates of HGV. This information can be used by one skilled inthe art to isolate new stains/isolates using the techniques ofhybridization, primer extension, and RT-PCR as described herein (e.g.,using degenerate primers based on the disclosed HGV variant sequences).

In the present case, HGV is an new isolate believed to be a member ofthe family Flaviviridae. Within this virus family, examination of thestructural proteins encoded by a virus allows the most definitivedetermination of whether a viral isolate is a member of a distinctspecies of virus. Non-structural proteins are most conserved betweendifferent species of viruses within a family of virus species. This isbelieved to be the result of the necessity for preserving enzymaticfunctions, such as, the following: the proteolytic cleavage of a viralpolyprotein, and replication of the RNA genome by viral helicase and RNAdependent RNA polymerase of the virus.

Examination of several species within any genus of the Flaviviridaefamily, e.g., the flavivirus genus, demonstrates that the genes forthese conserved functions are more highly conserved between species thanthe structural proteins. Accordingly, one of the major determiningfactors of whether a virus isolate represents a new species, versus a"variant isolate" of a known species, is a determination of globalhomology of the structural proteins between known viral species and thenew virus isolate.

Local homologies found within regions about 200 amino acids or lesswhich are found in non-structural proteins are indeterminant indicatorsof whether an isolate is a variant or a new species. Typically, virusisolates having global structural protein homologies of less than orabout 40% are classified as either different species (viruses) ordifferent genuses. The structural regions of HGV each have homologieslower than 40% compared with any virus described in "GENBANK"(comparisons carried out by methods standard in the art). Accordingly,HGV is considered to be a new species and possibly a new genus ofpositive strand RNA virus.

Another important region that is examined in determining thephylogenetic placement of a viral isolate is the 5' and 3' untranslatedregions (UTRs). These regions are compared between viral isolates. Forexample, all the members HCV, an unclassified genus of Flaviviridae,have 5' untranslated regions that are greater than about 90% conservedwith all other members in the genus. Further, the members of the HCVshare 3' untranslated regions between about 24 and about 50 nucleotideslong.

No significant alignments are found with any virus in "GENBANK" (Ver.86) when the 5'-untranslated region is used as a query sequence withFASTA on BLASTN. Further, HGV contains a 3' untranslated region that isat least about 250 nucleotides long that also contains little homologyto any other known virus.

Members of the Flaviviridae family are known to replicate in a widevariety of animals ranging from (i) hematophagous arthropod vectors(ticks and mosquitoes), where they do not cause disease, to (ii) a largerange of vertebrate hosts (humans, primates, other mammals, marsupials,and birds). Over 30 members of the Flaviviridae family cause diseases inman, ranging from febrile illness, or rash, to potentially fataldiseases such as hemorrhagic fever, encephalitis, or hepatitis. At least10 members of the Flaviviridae family cause severe and economicallyimportant diseases in domestic animals.

VI. Utility

A. The Invention.

In one aspect, the invention pertains to polynucleotides derived from aHepatitis G Virus (HGV) polynucleotide in substantially isolated form.In one embodiment the HGV polynucleotide is characterized by (i)transmission in primates, (ii) serologically distinguishable fromhepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus(HCV), hepatitis D virus, and hepatitis E virus (HEV), and (iii)membership of the virus family Flaviviridae. Polynucleotides of theinvention may be comprised of DNA or RNA (or analogs or variantsthereof) and may be produced recombinantly, isolated, or synthesizedaccording to methods known in the art.

Generally, HGV polynucleotides of the invention will be at least 10nucleotides in length. In an alternative embodiment, the HGVpolynucleotide will be at least 15 nucleotides in length. In still afurther alternative embodiment, the HGV polynucleotide will be at least20 nucleotides in length.

In a more specific embodiment, polynucleotides of the invention includecDNA or cDNA complements of the HGV genome. In a more specificembodiment, such a CDNA or cDNA complement will have at least a 40%sequence homology to a polynucleotide selected from the group consistingof SEQ ID NO:14, SEQ ID NO:37, and SEQ ID NO:19, or complements thereof.In yet another embodiment such cDNA's will exhibit at least 55% sequencehomology to a polynucleotide selected from the group consisting of SEQID NO:14, SEQ ID NO:37, and SEQ ID NO:19, or complements thereof. Inmore specific embodiments, cDNA or cDNA complement polynucleotides ofthe invention will have sequences derived from sequences selected fromthe group consisting of SEQ ID NO:14, SEQ ID NO:37, and SEQ ID NO:9, orcomplements thereof.

In another general embodiment, polynucleotides of the invention arepolynucleotide probes that specifically hybridize with HGV. In yetanother general embodiment, polynucleotides of the invention will encodean epitope of HGV. More specifically, such epitope encodingpolynucleotides may include sequences derived from SEQ ID NO:14, SEQ IDNO:19 or SEQ ID NO:37.

In another general embodiment, the polynucleotide of the inventionincludes a contiguous sequence of nucleotides that is capable ofselectively hybridizing to an HGV polynucleotide. In this regard, HGV ischaracterized as a genome comprising an open reading frame (ORF)encoding an amino acid sequence having at least 40% sequence homology toone of the following amino acid sequences: the 2873 amino acid sequenceof SEQ ID NO:15, the 190 amino acid sequence of SEQ ID NO:38, or the 67amino acid sequence of SEQ ID NO:20. More particularly, thepolynucleotide probe will specifically hybridize with HGV. Such apolynucleotide probe may carry detection labels or other modificationsor be fixed to a solid support.

DNA polynucleotides as described above may also encode an HGVspecifically immunoreactive antigenic determinants. In this regard, HGVis characterized as having a genome, cDNA or complements thereofcomprising an open reading frame (ORF) encoding an amino acid sequence.Such, an amino acid sequence having at least 40% sequence homology toone of the following amino acid sequences: the 2873 amino acid sequenceof SEQ ID NO:15, the 190 amino acid sequence of SEQ ID NO:38, or the 67amino acid sequence of SEQ ID NO:20.

In another specific embodiment, an HGV-encoding DNA polynucleotide thatis specifically reactive with an HGV antigenic determinant will, inaccordance with the invention, include an amino acid sequence having atleast 55% sequence homology to the 2873 amino acid sequence of SEQ IDNO:15 or to the 190 amino acid sequence of SEQ ID NO:38 or to the 67amino acid sequence of SEQ ID NO:20.

In yet another specific embodiment, the DNA polynucleotide may exhibitat least 40% sequence homology to a polynucleotide selected from thegroup consisting of SEQ ID NO:14, SEQ ID NO:37, and SEQ ID NO:19, orcomplements thereof.

In still a further embodiment, the invention includes a DNApolynucleotide that encodes an HGV-derived polypeptide. Moreparticularly, the polypeptide encoded by the polynucleotide will includea contiguous sequence of at least 15-60 amino acids having 55% sequencehomology to a contiguous sequence of at least 15-60 amino acids encodedby an HGV genome, cDNA or complements thereof.

In a specific embodiment, HGV-polypeptide encoding polynucleotides maybe encoded within the PNF 2161 cDNA source lambda gt11 library. In yetanother specific embodiment, the DNA polynucleotide may encode anepitope of HGV. In still a further embodiment, the polynucleotide may bea probe that specifically hybridizes with HGV.

In a related aspect, the invention includes a recombinant vector thatcontains a DNA polynucleotide that encodes an HGV polypeptide. Inanother related aspect, the invention includes a cell transformed withsuch a vector.

In still another related aspect, the invention includes a polynucleotideprobe that specifically hybridizes with an HGV hepatitis virus genome,CDNA or complements thereof. In a more specific embodiment, thepolynucleotide probe sequence has at least 40% homology to a sequencederived from SEQ ID NO:19, SEQ ID NO:37, or SEQ ID NO:14, or complementsthereof. In another specific embodiment, the polynucleotide probe isderived from SEQ ID NO:19, SEQ ID NO:37, or SEQ ID NO:14, or complementsthereof.

In another related aspect, the invention includes a method of detectingan HGV hepatitis virus nucleic acid in a test subject. According to themethod a nucleic acid-containing sample is obtained from the subject.The sample is then combined with and at least one polynucleotide probethat specifically hybridizes with the HGV hepatitis viral genome. HGVnucleic acid/probe complexes, formed by hybridization of the HGV nucleicacid with probe, are then detected. Such detecting may be accomplishedby hybridization of a probe containing at least one reporter moiety tothe HGV nucleic acid.

In a more specific embodiment, the above-described method includes theuse of HGV nucleic acid specific probes where the two probes (primers)define an internal region of the HGV nucleic acid. In this embodiment,each probe has one strand containing a 3'-end internal to the HGVnucleic acid internal region. The nucleic acid/probe hybridizationcomplexes are then converted to double-strand probe containing fragmentsby primer extension reactions. Probe-containing fragments are amplifiedby successively repeating the steps of (i) denaturing the double-strandfragments to produce single-strand fragments, (ii) hybridizing thesingle strands with the probes to form strand/probe complexes, (iii)generating double-strand fragments from the strand/probe complexes inthe presence of DNA polymerase and all four deoxyribonucleotides, and(iv) repeating steps (i) to (iii) until a desired degree ofamplification has been achieved. Amplification products are thenidentified according to established procedures. The method of theinvention may further include a third polynucleotide probe capable ofselectively hybridizing to the internal region described above but notto the specific probe/primer sequences used for amplification.

In another specific embodiment, detection of HGV nucleic acid/probecomplexes is accomplished by a target amplification method, such as byself-sustained sequence replication, ligase chain reaction, or stranddisplacement amplification. In a further specific embodiment detectionis accomplished employing a signal amplification technique such asbranch-chained DNA probes or the Q-beta replicase method.

In still another related aspect, the invention includes a kit foranalyzing samples for the presence of polynucleotides derived HGVhepatitis virus. In a general embodiment, the kit includes at least onepolynucleotide probe containing a nucleotide sequence that willspecifically hybridize with an HGV polynucleotide and a suitablecontainer. In a specific embodiment, the kit includes two polynucleotideprobes defining an internal region of the HGV polynucleotide, where eachprobe has one strand containing a 3'-end internal to the region. In afurther embodiment, the probes may be useful as primers for polymerasechain reaction amplification.

In still a further related aspect, the invention includes the HGVhepatitis virus particle in substantially isolated form. The inventionalso includes a polypeptide or a preparation of polypeptides from theHGV hepatitis virus in substantially isolated form. In this regard, theHGV virus is characterized as follows: (i) it is transmissible inprimates; (ii) it is serologically distinct from hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis Dvirus, and hepatitis E virus (HEV); and (iii) it is a member of thevirus family Flaviviridae. HGV polypeptides, as defined above, may beprepared by conventional means, including chemical synthesis andrecombinant DNA expression. Such polypeptides may also be fixed to asolid phase.

In a specific embodiment the polypeptide is specifically immunoreactivewith at least one anti-HGV antibody. In still a further specificembodiment, the polypeptide comprises an antigenic determinantspecifically immunoreactive with HGV. In this context, HGV ischaracterized by having a genome comprising an open reading frame (ORF)encoding an amino acid sequence having at least 40% sequence homology tothe 2873 amino acid sequence of SEQ ID NO:15 or to the 190 amino acidsequence of SEQ ID NO:38 or to the 67 amino acid sequence of SEQ IDNO:20. In a more specific embodiment, the ORF encodes amino acidsequence has at least 55% sequence homology to one of the aforementionedamino acid sequences. In still a further embodiment, the polypeptidesequence is derived from the 2873 amino acid sequence of SEQ ID NO:15,or fragments thereof, the 190 amino acid sequence of SEQ ID NO:38, orfragments thereof, or the 67 amino acid sequence of SEQ ID NO:20, orfragments thereof.

In another specific embodiment, the polypeptide from the HGV hepatitisvirus includes a contiguous sequence of at least about 60 amino acidsencoded by an HGV genome, cDNA or complements thereof. Morespecifically, such peptide sequence may be encoded by the PNF 2161 cDNAsource lambda gt11 library.

Recombinantly expressed HGV polypeptides may, in a more specificembodiment, include a polypeptide sequence derived from SEQ ID NO:20,SEQ ID NO:38, or SEQ ID NO:15. In another embodiment such a polypeptidemay be encoded by a sequence derived from SEQ ID NO:14, or from thecomplement of SEQ ID NO:14.

In a further related embodiment, in accordance with the invention, anHGV hepatitis virus polypeptide may be a fusion polypeptide comprisingan HGV polypeptide and a second polypeptide. More specifically, such afusion polypeptide may include, as a second polypeptide signalsequences, β-galactosidase or glutathione-S-transferase proteinsequences. Alternatively, the second polypeptide may comprise a particleforming protein.

The above-described polypeptides may be derived from structural ornon-structural viral proteins.

In still a further related aspect, the invention includes a cloningvector capable of expressing, under suitable conditions, an open readingframe (ORF) of cDNA derived from HGV hepatitis virus genome, cDNA orcomplements thereof. In this aspect of the invention, the ORF isoperably linked to a control sequence compatible with a desired host. Ina related aspect, the invention includes a cell transformed with such avector. In a more specific embodiment of the vector, the ORF may bederived from SEQ ID NO:14 or its complement. In yet further specificembodiments, the ORF may be derived from SEQ ID NO:37 or SEQ ID NO:19.

In a related aspect, the invention includes a method of producing an HGVhepatitis virus polypeptide. The method includes culturing cellscontaining the above-described vectors under conditions suitable toachieve expression of the open reading frame (ORF) sequence. In a morespecific embodiment, the ORF sequence encodes a polypeptide sequenceselected from the group of polypeptide sequences, or fragments thereof,consisting of SEQ ID NO:15, SEQ ID NO:38 and SEQ ID NO:20. Further, theORF sequences may be derived from an HGV cDNA, or complement thereof. Inyet another specific embodiment, the vector is a lambda gt11 phagevector expressed in Escherichia coli cells.

In a further related aspect, the invention includes a diagnostic kit foruse in screening serum containing antibodies specific against HGVhepatitis virus infection. Such a kit may include a substantiallyisolated HGV polypeptide antigen comprising an epitope which isspecifically immunoreactive with at least one anti-HGV antibody. Such akit also includes means for detecting the binding of said antibody tothe antigen. In regard to such a kit, HGV is characterized by having agenome, cDNA or complements thereof comprising an open reading frame(ORF) encoding an amino acid sequence. Such an amino acid sequencetypically having at least 40% sequence homology to the 2873 amino acidsequence of SEQ ID NO:15 or to the 190 amino acid sequence of SEQ IDNO:38 or to the 67 amino acid sequence of SEQ ID NO:20. In specificembodiments, the kit may include a recombinantly produced or chemicallysynthesized polypeptide antigen. The polypeptide antigen of the kit mayalso be attached to a solid support.

In a more specific embodiment, the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labelled anti-human antibody. In this embodiment, binding ofthe antibody to the HGV polypeptide antigen can be detected by bindingof the reporter-labelled antibody the antibody.

In a related aspect, the invention includes a method of detecting HGVhepatitis virus infection in a test subject. This detection methodincludes reacting serum from an HGV test subject with a substantiallyisolated HGV polypeptide antigen, and examining the antigen for thepresence of bound antibody. In a specific embodiment, the methodincludes a polypeptide antigen attached to a solid support, and theserum is reacted with the support. Subsequently, the support is reactedwith a reporter-labelled anti-human antibody. The solid support is thenexamined for the presence of reporter-labelled antibody.

In a further aspect, the invention includes an HGV hepatitis virusvaccine composition. The composition includes a substantially isolatedHGV polypeptide antigen, where the antigen includes an epitope which isspecifically immunoreactive with at least one anti-HGV antibody. Thepeptide antigen may be produced according to methods known in the art,including recombinant expression or chemical synthesis. The peptideantigen is preferably present in a pharmacologically effective dose in apharmaceutically acceptable carrier.

In still a further related aspect, the invention includes a monoclonalantibody that is specifically immunoreactive with the HGV hepatitisvirus epitope. In another related aspect, the invention includes asubstantially isolated preparation of polyclonal antibodies specificallyimmunoreactive with HGV. In a more specific embodiment, such polyclonalantibodies are prepared by affinity chromatography.

In a related aspect, the invention includes a method for producingantibodies to HGV. The method includes administering to a test subject asubstantially isolated HGV polypeptide antigen, where the antigenincludes an epitope which is specifically immunoreactive with at leastone anti-HGV antibody. The antigen is administered in an amountsufficient to produce an immune response in the subject.

In yet another related aspect, the invention includes a diagnostic kitfor use in screening serum containing HGV antigens. The diagnostic kitincludes a substantially isolated antibody specifically immunoreactivewith an HGV polypeptide antigen, and means for detecting the binding ofthe polypeptide antigen to the antibody. In one embodiment, the antibodyis attached to a solid support. In a specific embodiment, the antibodymay be a monoclonal antibody. The detecting means of the kit may includea second, labelled monoclonal antibody. Alternatively, or in addition,the detecting means may include a labelled, competing antigen.

In another, related aspect, the invention includes a method of detectingHGV infection in a test subject. According to this aspect of theinvention, serum from a test subject is reacted with a substantiallyisolated HGV specific antibody of the kit described above. The HGVspecific antibody is then examined for the presence of bound antigen.

In still a further related aspect, the invention includes an in vitrogrown cell infected with HGV. In a specific embodiment, the cell is ahepatocyte grown in tissue culture. More specifically, the tissueculture cell may be an immortalized hepatocyte, or it may be a from acell line derived from liver of an HGV infected primate.

In a related aspect, the invention includes a method of propagating HGV.The method includes culturing in vitro grown, HGV-infected cells, asdescribed above, under conditions effective to promote the propagationof HGV. In another related aspect, the invention includes HGV particlesproduced by such a propagation method.

In still a further aspect, the invention includes a mosaic polypeptide.Such a polypeptide may include at least two epitopes of HGV, where thepolypeptide substantially lacks amino acids normally intervening betweenthe epitopes in the native HGV coding sequence. In a more specificembodiment, the mosaic polypeptide is attached to a solid support. Instill a further related aspect, the invention includes a nucleic acidthat encodes the above-described mosaic polypeptide.

In another related aspect, the invention includes a method of detectingHGV infection in a test subject. The method includes contacting anantibody-containing sample from the subject with a mosaic polypeptide,as described above, and examining the antigen for the presence of boundantibody.

In still a further related aspect, the invention includes an HGV vaccinecomposition. The vaccine composition includes mosaic polypeptide thatincludes more than one HGV epitope. The mosaic polypeptide is present ina pharmacologically effective dose in a pharmaceutically acceptablecarrier.

B. Immunoassays for HGV.

One utility for the antigens obtained by the methods of the presentinvention is their use as diagnostic reagents for the detection ofantibodies present in the sera of test subjects infected with HGVhepatitis virus, thereby indicating infection in the subject; forexample, 470-20-1 antigen, antigens encoded by SEQ ID NO:14 or itscomplement, and antigens encoded by portions of either strand of thecomplete viral sequence. The antigens of the present invention can beused singly, or in combination with each other, in order to detect HGV.The antigens of the present invention may also be coupled withdiagnostic assays for other hepatitis agents such as HAV, HBV, HCV, andHEV.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention, e.g., the 470-20-1 antigen. After binding withanti-HGV antibody to the reagent and removing unbound serum componentsby washing, the reagent is reacted with reporter-labelled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-HGV antibody on the solid support. The reagent is againwashed to remove unbound labelled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric or calorimetric substrate (Sigma, St. Louis,Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Also forming part of the invention is an assay system or kit forcarrying out this diagnostic method. The kit generally includes asupport with surface-bound recombinant HGV antigen (e.g., the 470-20-1antigen, as above), and a reporter-labelled anti-human antibody fordetecting surface-bound anti-HGV antigen antibody.

In a second diagnostic configuration, known as a homogeneous assay,antibody binding to a solid support produces some change in the reactionmedium which can be directly detected in the medium. Known general typesof homogeneous assays proposed heretofore include (a) spin-labelledreporters, where antibody binding to the antigen is detected by a changein reported mobility (broadening of the spin splitting peaks), (b)fluorescent reporters, where binding is detected by a change influorescence efficiency or polarization, (c) enzyme reporters, whereantibody binding causes enzyme/substrate interactions, and (d)liposome-bound reporters, where binding leads to liposome lysis andrelease of encapsulated reporter. The adaptation of these methods to theprotein antigen of the present invention follows conventional methodsfor preparing homogeneous assay reagents.

In each of the assays described above, the assay method involvesreacting the serum from a test individual with the protein antigen andexamining the antigen for the presence of bound antibody. The examiningmay involve attaching a labelled anti-human antibody to the antibodybeing examined (for example from acute, chronic or convalescent phase)and measuring the amount of reporter bound to the solid support, as inthe first method, or may involve observing the effect of antibodybinding on a homogeneous assay reagent, as in the second method.

A third diagnostic configuration involves use of HGV antibodies capableof detecting HGV-specific antigens. The HGV antigens may be detected,for example, using an antigen capture assay where HGV antigens presentin candidate serum samples are reacted with a HGV specific monoclonal orpolyclonal antibody. The antibody is bound to a solid substrate and theantigen is then detected by a second, different labelled anti-HGVantibody. Antibodies can be prepared, utilizing the peptides of thepresent invention, by standard methods. Further, substantially isolatedantibodies (essentially free of serum proteins which may affectreactivity) can be generated (e.g., affinity purification (Harlow etal.)).

C. Hybridization Assays for HGV.

One utility for the nucleic acid sequences obtained by the methods ofthe present invention is their use as diagnostic agents for HGVsequences present in sera, thereby indicating infection in theindividual. Primers and/or probes derived from the coding sequences ofthe present invention, in particular, Clone 470-20-1 and SEQ ID NO:14,can be used singly, or in combination with each other, in order todetect HGV.

In one diagnostic configuration, test serum is reacted under PCR orRT-PCR conditions using primers derived from, for example, 470-20-1sequences. The presence of HGV, in the serum used in the amplificationreaction, can be detected by specific amplification of the sequencestargeted by the primers. Example 4 describes the use of polymerase chainamplification reactions, employing primers derived from the clones ofthe present invention, to screen different source material. The resultsof these amplification reactions demonstrate the ability of primersderived from the clones of the present invention (for example,470-20-1), to detect homologous sequences by amplification reactionsemploying a variety of different source templates. The amplificationreactions in Example 4 included use of nucleic acids obtained directlyfrom sera as template material.

Alternatively, probes can be derived from the HGV sequences of thepresent invention. These probes can then be labelled and used ashybridization probes against nucleic acids obtained from test serum ortissue samples. The probes can be labelled using a variety of reportermolecules and detected accordingly: for example, radioactive isotopiclabelling and chemiluminescent detection reporter systems (Tropix,Bedford, Mass.).

Target amplification methods, embodied by the polymerase chain reaction,the self-sustained sequence replication technique "3SR," (Guatelli, etal.; Gingeras, et al., 1990) also known as "NASBA" (VanGemen, et al.)!,the ligase chain reaction (Barany), strand-displacement amplification"SDA," (Walker)!, and other techniques, multiply the number of copies ofthe target sequence. Signal amplification techniques, exemplified bybranched-chain DNA probes (Horn and Urdea; Urdea; Urdea, et al.) and theQ-beta replicase method (Cahill, et al.; Lomell, et al.), first bind aspecific molecular probe, then replicate all of or part of this probe orin some other manner amplify the probe signal.

For the detection of the specific nucleic acid sequences disclosed inthe present invention or contiguous sequences in the same or a similar(related) viral genome, amplification and detection methodologies may beemployed, as alternatives to amplification by the PCR. A number of suchtechniques are known to the field of nucleic acid diagnostics (The 1992San Diego Conference: Genetic Recognition, Clin. Chem. 39(4):705(1993)).

1. SELF-SUSTAINED SEQUENCE REPLICATION.

The Self-Sustained Sequence Replication (3SR) technique results inamplification to a similar magnitude as PCR, but isothermally. Ratherthan thermal cycle-driven PCR, the 3SR operates as a concertedthree-enzyme reaction of a) CDNA synthesis by reverse transcriptase, b)RNA strand degradation by RNase H, and c) RNA transcription by T7 RNApolymerase.

As the entire reaction sequence occurs isothermally (typically at 42°C.), expensive temperature-cycling instrumentation is not required. Inthe absence of duplex denaturation via heating, organic solvents, orother mechanism, only single-stranded templates (i.e., predominantlyRNA) are amplified.

Suitable primers for use in 3SR amplification can be selected from theviral sequences of the present invention by those having ordinary skillin the art. For example, for isothermal amplification of viral sequencesby the 3SR technique, primer 470-20-1-77F (SEQ ID NO:9) is modified bythe addition of the T7 promoter sequence and a preferred T7transcription initiation site to the 5'-end of the oligonucleotide. Thismodification results in a suitable 3SR primer T7-470-20-1-77F (SEQ IDNO:9). Primer 470-20-1-211R (SEQ ID NO:10) can be used in thesereactions either without modification or T7 promoter.

RNA extracted from PNF 2161 is incubated with AMV reverse transcriptase(30 U), RNase H (3 U), T7 RNA polymerase (100 U), in 100 ul reactionscontaining 20 mM Tris-HCl, pH 8.1 (at room temperature), 15 mM MgCl₂, 10mM KC1, 2 mM spermidine HC1, 5 mM dithiothreitol (DTT), 1 mM each ofdATP, dCTP, dGTP, and TTP, 7 mM each of ATP, CTP, GTP, and UTP, and 0.15uM each primer. Amplification takes place during incubation at 42° C.for 1-2 h.

Initially, primer T7-470-20-1-77F anneals to the target RNA, and isextended by AMV reverse transcriptase to form CDNA complementary to thestarting RNA strand. Following degradation of the RNA strand by RNase H,reverse transcriptase catalyzes the synthesis of the second strand DNA,resulting in a double-stranded template containing the (double-stranded)T7 promoter sequence. RNA transcription results in production ofsingle-stranded RNA. This RNA then serves to re-enter the cycle foradditional rounds of amplification, finally resulting in a pool ofhigh-concentration product RNA. The product is predominantlysingle-stranded RNA of the same strand as the primer containing the T7promoter (T7-470-20-1-77F), with much smaller amounts of cDNA.

Alternatively, the other primer (470-20-1-211R) may contain the T7promoter, or both primers may contain the promoter, resulting inproduction of both strands of RNA as products of the reaction. Productsof the 3SR reaction may be detected, characterized, or quantitated bystandard techniques for the analysis of RNA (e.g., Northern blots, RNAslot or dot blots, direct gel electrophoresis with RNA-staining dyes).Further, the products may be detected by methods making use ofbiotin-avidin affinity interactions or specific hybridizations ofnucleic acid probes.

In one technique for rapid and specific analysis of 3SR products,solution hybridization of the product to radiolabelled oligonucleotide470-20-1-152R (SEQ ID NO:21) is followed by non-denaturingpolyacrylamide gel electrophoresis. This assay (a gel mobilityshift-type assay) results in the detection of specific probe-producthybrid as a slower-moving band than the band corresponding tounhybridized oligonucleotide.

2. LIGASE CHAIN REACTION (LCR)

As another example of a detection system, the HGV sequence may form thebasis for design of ligase chain reaction (LCR) primers. LCR makes useof the nick-closing activity of DNA ligase to join two immediatelyadjacent oligonucleotides possessing adjacent 5'-phosphate ("donor"oligo) and 3'-hydroxyl ("¹ acceptor" oligo) terminii. The property ofDNA ligase to join only fully complementary ends in a template-dependentway, leads to a high degree of specificity, in that ligation will notoccur unless the terminii to be linked are perfectly matched in sequenceto the target strand.

As an alternative to PCR, with some advantages in terms of specificityfor discrimination of single base mismatches between primer and targetnucleic acid, the LCR may be used to detect or "type" strains of viruspossessing homology to HGV sequences. These techniques are suitable forassessing the presence of specific mutations when such base changes areknown to confer drug resistance (e.g., Larder and Kemp; Gingeras, etal., 1991). In the presence of template-complementary donor and acceptoroligonucleotides and oligonucleotides complementary to the donor andacceptor, exponential amplification by LCR is possible. In thisembodiment, each round of ligation generates additional template forsubsequent rounds, in a cyclic reaction.

For example, primer 470-20-1-211R (SEQ ID NO:10), an adjacentoligonucleotide (B, SEQ ID NO:22) and cognate oligos (211R', SEQ IDNO:23, and B', SEQ ID NO:24), can be used to perform LCR amplificationof the sequence of this invention. Reverse transcription is firstperformed by standard methods to generate cDNA, which is then amplifiedin reactions containing 0.1-1 μM each of the four LCR primers, 20 mMTris-HCl, pH 8.3 (room temperature), 25 mM KC1, 10 mM MgCl₂, 10 mMdithiothreitol (DTT), 0.5 mM AND+, 0.01% Triton X-100, and 5 Units ofDNA ligase (Ampligase, Epicentre Technologies, Madison, Wis., or othercommercial supplier of thermostable DNA ligase), in 25 ul reactions.

Thermal cycling is performed at 94° C. for 1 min. 30 s; 94° C. for 1min., 65° C. for 2 min., repeated for 25-40 cycles. Specificity ofproduct synthesis depends on primer-template match at the 3'-terminalposition. Products are detected by polyacrylamide gel electrophoresis,followed by ethidium bromide staining; alternatively, one of theacceptor oligos (211R' or B) is 5'-radiolabelled for visualization byautoradiography following gel electrophoresis.

Alternatively, a donor oligo is 3'-end-labelled with a specific bindablemoiety (e.g., biotin), and the acceptor is 5'-labelled with a specificdetectable group (e.g., a fluorescent dye), for solid phase capture anddetection.

3. METHODS FOR ANALYSIS OF AMPLIFIED DNA

Numerous techniques have been described for the analysis of amplifiedDNA. Several such techniques are advantageous for high-throughputapplications, where gel electrophoresis is impractical, for example,rapid and high-resolution HPLC techniques (Katz and Dong). However, ingeneral, methods for infectious disease organism screening using nucleicacid probes involve a separate post-amplification hybridization step inorder to assure requisite specificity for pathogen detection.

One such detection embodiment is an affinity-based hybrid capturetechnique (Holodniy, et al.). In this embodiment the PCR is conductedwith one biotinylated primer. Following amplification, thedouble-stranded product is denatured then hybridized to aperoxidase-labelled probe complementary to the strand havingincorporated the biotinylated primer. The hybridized product is thenincubated in a buffer which is in contact with an avidin (orstreptavidin) coated surface (e.g., membrane filter, microwell, latex orparamagnetic beads).

The mass of coated solid phase which contacts the volume of PCR productto be analyzed by this method must contain sufficient biotin-bindingsites to capture essentially all of the free biotinylated primer, aswell as the much lower concentration of biotinylated PCR product.Following three to four washes of the solid phase, bound hybridizedproduct is detected by incubation with o-phenylenediamine in citratebuffer containing hydrogen peroxide.

Alternatively, capture may be mediated by probe-coated surfaces,followed by affinity-based detection via the biotinylated primer and anavidin-reporter enzyme conjugate (Whetsell, et al.).

4. ADDITIONAL METHODS

Viral sequences of the present invention may also form the basis for asignal amplification approach to detection, using branched-chain DNAprobes. Branched-chain probes (Horn and Urdea; Urdea) have beendescribed for detection and quantification of rare RNA and DNA sequences(Urdea, et al.). In this method, an oligonucleotide probe (RNA, DNA, ornucleic acid analogue) is synthesized with a sequence complementary tothe target RNA or DNA. The probe also contains a unique branchingsequence or sequences not complementary to the target RNA or DNA.

This unique sequence constitutes a target for hybridization of branchedsecondary detector probes, each of which contains one or more otherunique sequences, serving as targets for tertiary probes. At each branchpoint in the signal amplification pathway, a different unique sequencedirects hybridization of secondary, tertiary, etc., detection probes.The last probe in the series typically is linked to an enzyme useful fordetection (e.g., alkaline phosphatase). The sequential hybridization ofprimers eventually results in the buildup of a highly-branchedstructure, the arms of which terminate in enzyme-linked probes.

Enzymatic turnover provides a final amplification, and the choice ofhighly sensitive chemiluminescent substrates (e.g., LumiPhos, Lumigen,Detroit, Mich., as a substrate for alkaline phosphatase labels) resultsin exquisite sensitivity, on the order of 10,000 molecules or less oforiginal target sequence per assay. In such a detection method,amplification depends only on molecular hybridization, rather thanenzymatic mechanisms, and is thus far less susceptible to inhibitorysubstances in clinical specimens than, for example, PCR. Thus, thisdetection method allows the use of crude techniques for nucleic acidrelease in test samples, without extensive purification before assay.

Amplification for sensitive detection of the viral sequences of thepresent invention may also be accomplished by the Q-β replicasetechnique (Cahill, et al.; Lomell, et al.; Pritchard, et al.). In thismethod, a specific probe is designed to be complementary to the targetsequence. This probe is then inserted by standard molecular cloningtechniques into the sequence of the replicatable RNA from Q-β phage.Insertion into a specific region of the replicon does not preventreplication by Q-β replicase.

Following molecular hybridization, and several cycles of washing, thereplicase is added and amplification of the probe RNA ensues."Reversible target capture" is one known technique for reducing thepotential background from replication of unhybridized probes (Morrissey,et al.). Amplified replicons are detectable by standard molecularhybridization techniques employing DNA, RNA or nucleic acid analogueprobes.

Additional methods for amplification and detection of rare DNA or RNAsequences are known in the literature and preferred to the PCR for someapplications in the field of molecular diagnostics. These alternativetechniques may form the basis for detection, characterization (e.g.,sequence diversity existing as multiple related strains of the sequencedescribed herein, genotypic changes characteristic of drug resistance),or quantification of the sequence disclosed in the present invention.

Also forming part of the invention are assay systems or kits forcarrying out the amplification/-hybridization assay methods justdescribed. Such kits generally include either specific primers for usein amplification reactions or hybridization probes.

D. Therapeutic Uses.

As discussed above, the HGV antigens of the present invention can beused in vaccine preparation.

Further, antibodies generated against the polypeptide antigens of thepresent invention can be used for passive immunotherapy or passiveimmunoprophylaxis. The antibodies can be administered in amounts similarto those used for other therapeutic administrations of antibody. Forexample, pooled gamma globulin is administered at 0.02-0.1 ml/lb bodyweight during the early incubation of other viral diseases such asrabies, measles and hepatitis B to interfere with establishment ofinfection. Thus, antibodies reactive with the HGV antigens can bepassively administered alone or in conjunction with another anti-viralagent to a host infected with HGV to enhance the ability of the host todeal with the infection.

The HGV sequences disclosed herein identify HGV as a member of theFlaviviridae family (see above). The Flaviviridae are classified into 3genera, flaviviruses, petstiviruses, and the hepatitis C virus genera(Francki, et al.). All Flaviviridae possess a positive strand RNA genomeof 9.0-12 kb in length which encodes a single long polypeptide of3000-4000 amino acids. This polypeptide is proteolytically cleaved intoapproximately 10 proteins, including, a viral capsid protein, viralenvelope protein(s), and a minimum of 5 non-structural proteins (NS).The non-structural proteins include a chymotrypsin like serine protease,RNA helicase (NS3), and an RNA-dependent RNA polymerase (NS5). The NS3protein of Flaviviridae is required for proteolytic cleavage of theviral polypeptide. The NS5 protein is required for replication of theviral genome (Chambers, et al., 1990a).

Additionally, several cellular proteins have been identified as beinginvolved in the replication of the Flaviviridae. For example, cellularsignal peptidase enzyme may be required to cleave the viral polypeptideat several cleavage sites, to allow for expression of the viral protease(Hijikata, et al.).

Inhibitors which prevent these proteins from carrying out their requiredfunctions in flavivirus replication may also have therapeutic value attreating infection with HGV. Finally cytokines or other polypeptideswhich are known to have antiviral activity and/or modulate the humanimmune system may be efficacious at treating HGV infection.

One compound known to inhibit Flaviviridae RNA dependent RNApolymerases, which by analogy may be expected to inhibit the activity ofthe NS5 protein of HGV, is the nucleotide analogue1-B-D-ribofuranosyl-1-2,4-triazole, 3-carboxamide, also known asribavirin (Patterson, et al.). The method of action of ribavirin isthought to involve depletion of intercellular guanine pools andinterference with the capping of viral RNAs (Patterson et al.).

In individuals infected with HCV, significant reductions in viral titerand in serum levels of alanine aminotransferase (ALT--an indicatorenzyme for liver dysfunction) were observed while ribavirin wasadministered (Reichard, et al.; Di Bisceglie, et al., 1992). Ribavirinappears to have broad efficacy for treating Flaviviridae infections,accordingly, beneficial results are expected after administration ofribavirin to individuals suffering from HGV derived liver disease.

Another class of compounds known to be efficacious for treatingFlaviviridae infections include the cytokines interferon α, interferonβ, and interferon γ (Baron, et al.; Gutterman). Interferons are thoughtto act as antivirals by both (i) inducing the expression of cellularproteins that interfere with the replication and translation of viralRNAs, and (ii) by the activation of components of the human cellularimmune system (Baron, et al.) . The interferons have broad applicabilityto the treatment of viral infections including infection with HBV, HDV,and HCV (Gutterman; Farci, et al.). In particular, multiple studies haveindicated that the interferons, either alone or in combination withother antiviral therapies, are effective at treating infection withhepatitis C virus (Di Bisceglie, et al., 1989; Kakumu, et al.). Due toboth the apparent hepatotropic nature of HGV and its classification inthe family Flaviviridae, HGV infection may be expected to respond tosimilar interferon therapy.

Still another class of compounds with potent anti-viral activity areinhibitors of viral proteases (Krausslich, et al.). All Flaviviridaeencode a chymotrypsin-like serine protease which is required to cleavemultiple sites of the genome polypeptide at multiple sites in thenon-structural region. The amino acid residues that make up thecatalytic site of this protease are well described and include aHistidine, an Aspartic acid, and a Serine residue (Grakoui, et al.).Furthermore studies of the flavivirus, Yellow Fever Virus have indicatedthat mutation of the Serine residue of the active site inhibits viralreplication (Chambers, et al., 1990b).

Inhibitors of the HGV NS3 protein can be designed to mimic thetransition state of enzymatic cleavage. Alternatively, such inhibitorsmay be isolated by mass screening of previously synthesized compounds.The activity of putative HGV NS3 proteinase inhibitors can be determinedthrough the use of in vitro transcription/translation systems, which arewidely used in Flaviviridae research (Hijikata, et al.; Grakoui, etal.).

Alternatively, the HGV genome can be cloned into a suitable vector foreukaryotic protein expression, such a bacculovirus or vaccinia, and theefficacy of the compounds can be determined in tissue culture systems(Grakoui, et al.). Similar approaches have been employed successfully toobtain potent inhibitors of the HIV protease (Vacca, et al.; Roberts, etal.).

Another approach to treating disease caused by infection with the HGVrelies on the synthesis of antisense oligonucleotides (Tonkinson andStein) or oligonucleotide analogs which encode portions of the sequencesof HGV disclosed in the present invention. As is true for allFlaviviridae, it would be expected that the genome of HGV is a positivestrand RNA molecule of 9-12 kb in size. The single stranded nature ofthe viral genome should make HGV exquisitely sensitive to antisenseoligonucleotides. Possible target sequences which might be employed toinhibit viral replication include the 5' untranslated region of HGV, theribosome binding site of HGV or other sequences which would interferewith the translation of the HGV genome.

Antisense oligonucleotides can be synthesized using commerciallyavailable synthesizers. Preferably the oligonucleotides are synthesizedusing phosphorodithioate backbones which have the advantage of beingresistant to nuclease cleavage (Marshall & Caruthers). Additionallyother oligonucleotide analogues, such as those having a uncharged oramide type backbone (Egholm, et al.) may be employed. Theseoligonucleotides are commercially available (Biosearch, Millipore,Bedford, Mass.) and advantageous in that their lack of charge allowsthem to cross biological membranes, which are typically resist thepassage of charged macromolecules.

Oligonucleotides (or analogs thereof) for antisense applications aretypically greater than 8 nucleotides in length to facilitatehybridization to a target sequence within the HGV genome. Uponhybridization of, for example, DNA oligomers to viral RNA targetsequences, the hybridization complex can be degraded by a cellularenzyme such as RNAse H. The reduction in HGV templates then lessens theseverity of HGV associated disease.

The usefulness and efficacy of the above described therapeutic methodscan be evaluated in vitro, using the cell systems described above, andin vivo, using the animal model systems described above.

The following examples illustrate, but in no way are intended to limitthe present invention.

MATERIALS AND METHODS

Synthetic oligonucleotide linkers and primers were prepared usingcommercially available automated oligo-nucleotide synthesizers.Alternatively, custom designed synthetic oligonucleotides may bepurchased from commercial suppliers.

Standard molecular biology and cloning techniques were performedessentially as previously described in Ausubel, et al., Sambrook, etal., and Maniatis, et al.

Common manipulations relevant to employing antisera and/or antibodiesfor screening and detection of immunoreactive protein antigens wereperformed essentially as described (Harlow, et al.). Similarly ELISA andWestern blot assays for the detection of anti viral antibodies wereperformed either as described by their manufacturer (Abbott, N. Chicago,Ill., Genelabs Diagnostics, Singapore) or using standard techniquesknown in the art (Harlow, et al).

EXAMPLES EXAMPLE 1 Construction of PNF2161 cDNA Libraries

A. Isolation of RNA from Sera.

One milliliter of undiluted PNF 2161 serum was precipitated by theaddition of PEG (MW 6,000) to 8% and centrifugation at 12K, for 15minutes in a microfuge, at 4° C. RNA was extracted from the resultingserum pellet essentially as described by Chomczynski.

The pellet was treated with a solution containing 4M guanidiniumisothiocyanate, 0.18% 2-mercaptoethanol, and 0.5% sarcosyl. The treatedpellet was extracted several times with acidic phenol-chloroform, andthe RNA was precipitated with ethanol. This solution was held at -70° C.for approximately 10 minutes and then spun in a microfuge at 4° C. for10 minutes. The resulting pellet was resuspended in 100 μl ofDEPC-treated (diethyl pyrocarbonate) water, and 10 μl of 3M NaOAc,pH=5.2, two volumes of 100% ethanol and one volume of 100% isopropanolwere added to the solution. The solution was held at -70° C. for atleast 10 minutes. The RNA pellet was recovered by centrifugation in amicrofuge at 12,000 ×g for 15 minutes at 5° C. The pellet was washed in70% ethanol and dried under vacuum.

B. SYNTHESIS OF cNA (i) FIRST STRAND SYNTHESIS

The synthesis of cDNA molecules was accomplished as follows. The abovedescribed RNA preparations were transcribed into cDNA, according to themethod of Gubler et al. using random nucleotide hexamer primers (CDNASynthesis Kit, BMB, Indianapolis, Ill. or GIBCO/BRL).

After the second-strand cDNA synthesis, T4 DNA polymerase was added tothe mixture to maximize the number of blunt-ends of cDNA molecules. Thereaction mixture was incubated at room temperature for 10 minutes. Thereaction mixture was extracted with phenol/chloroform and chloroformisoamyl alcohol.

The cDNA was precipitated by the addition of two volumes of 100% ethanoland chilling at -70° C. for 15 minutes. The cDNA was collected bycentrifugation, the pellet washed with 70% ethanol and dried undervacuum.

C. Amplification of the Double Stranded cDNA Molecules.

The cDNA pellet was resuspended in 12 μl distilled water. To theresuspended cDNA molecules the following components were added: 5 μlphosphorylated linkers (Linker AB, a double strand linker comprised ofSEQ ID NO:1 and SEQ ID NO:2, where SEQ ID NO:2 is in a 3' to 5'orientation relative to SEQ ID NO:1--as a partially complementarysequence to SEQ ID NO:l), 2 μl 10× ligation buffer (0.66M Tris.ClpH=7.6, 50 MM MgCl₂, 50 mM DTT, 10 mM ATP) and 1 μl T4 DNA ligase (0.3to 0.6 Weiss Units). Typically, the cDNA and linker were mixed at a1:100 ratio. The reaction was incubated at 14° C. overnight. Thefollowing morning the reaction was incubated at 70° C. for three minutesto inactivate the ligase.

To 100 μl of 10 mM Tris-Cl buffer, pH 8.3, containing 1.5 MM MgCl₂ and50 mM KC1 (Buffer A) was added about 1 μl of the linker-ligated cDNApreparation, 2 μM of a primer having the sequence shown as SEQ ID NO:1,200 AM each of DATP, dCTP, dGTP, and dTTP, and 2.5 units of Thermusaguaticus DNA polymerase (Taq polymerase). The reaction mixture washeated to 940° C. for 30 sec for denaturation, allowed to cool to 50° C.for 30 sec for primer annealing, and then heated to 72° C. for 0.5-3minutes to allow for primer extension by Taq polymerase. Theamplification reaction, involving successive heating, cooling, andpolymerase reaction, was repeated an additional 25-40 times with the aidof a Perkin-Elmer Cetus DNA thermal cycler (Mullis; Mullis, et al.;Reyes, et al., 1991; Perkin-Elmer Cetus, Norwalk, Conn.).

After the amplification reactions, the solution was thenphenol/chloroform, chloroform/isoamyl alcohol extracted and precipitatedwith two volumes of ethanol. The resulting amplified CDNA pellets wereresuspended in 20 Al TE (pH=7.5).

D. Cloning of the cDNA into Lambda Vectors.

The linkers used in the construction of the cDNAs contained an EcoRIsite which allowed for direct insertion of the amplified cDNAs intolambda gt11 vectors (Promega, Madison Wis. or Stratagene, La Jolla,Calif.). Lambda vectors were purchased from the manufacturer (Promega)which were already digested with EcoRI and treated with alkalinephosphatase, to remove the 5' phosphate and prevent self-ligation of thevector.

The EcoRI-digested cDNA preparations were ligated into lambda gt11(Promega). The conditions of the ligation reactions were as follows: 1μl vector DNA (Promega, 0.5 mg/ml); 0.5 or 3 μl of the PCR amplifiedinsert cDNA; 0.5 μl 10×ligation buffer (0.5M Tris-HCl, pH=7.8; 0.1MMgCl₂ ; 0.2 M DTT; 10 mM ATP; 0.5 mg/ml bovine serum albumin (BSA)), 0.5μl T4 DNA ligase (0.3 to 0.6 Weiss units) and distilled water to a finalreaction volume of 5 μl.

The ligation reactions were incubated at 14° C. overnight (12-18 hours).The ligated cDNA was packaged by standard procedures using a lambda DNApackaging system ("GIGAPAK", Stratagene, LaJolla, Calif.), and thenplated at various dilutions to determine the titer. A standard X-galblue/white assay was used to determine recombinant frequency of thelibraries (Miller; Maniatis et al.).

Percent recombination in each library was also determined as follows. Anumber of random clones were selected and corresponding phage DNAisolated. Polymerase chain reaction (Mullis; Mullis, et al.) was thenperformed using isolated phage DNA as template and lambda DNA sequences,derived from lambda sequences flanking the EcoRI insert site for thecDNA molecules, as primers. The presence or absence of insert wasevident from gel analysis of the polymerase chain reaction products.

The cDNA-insert phage libraries generated from serum sample PNF 2161 wasdeposited with the American Type Culture Collection, 12301 Parklawn Dr.,Rockville Md. 20852, and has been assigned the deposit designation ATCC75268 (PNF 2161 CDNA source).

EXAMPLE 2 Immunoscreening of Recombinant Libraries

The lambda gt11 libraries generated in Example 1 were immunoscreened forthe production of antigens recognizable by the PNF 2161 serum from whichthe libraries were generated. The phage were plated for plaque formationusing the Escherichia coli bacterial plating strain E. coli KM392.Alternatively, E. coli Yl09OR (Promega, Madison Wis.) may be used.

The fusion proteins expressed by the lambda gt11 clones were screenedwith serum antibodies essentially as described by Ausubel, et al.

Each library was plated at approximately 2×10⁴ phages per 150 mm plate.Plates were overlaid with nitrocellulose filters overnight. Filters werewashed with TBS (10 mM, Tris pH 7.5; 150 mM NaCl), blocked with AIB (TBSbuffer with 1% gelatin) and incubated with a primary antibody diluted100 times in AIB.

After washing with TBS, filters were incubated with a second antibody,goat-anti-human IgG conjugated to alkaline phosphatase (Promega).Reactive plaques were developed with a substrate (for example, BCIP,5-bromo-4-chloro-3-indolyl-phosphate), with NBT (nitro blue tetrazoliumsalt (Sigma)). Positive areas from the primary screening were replatedand immunoscreened until pure plaques were obtained.

EXAMPLE 3 Screening of the PNF 2161 Library

The cDNA library of PNF 2161 in lambda gt11 was screened, as describedin Example 2, with PNF 2161 sera. The results of the screening arepresented in Table 1.

                  TABLE 1                                                         ______________________________________                                        PNF2161 Libraries                                                                                                  # Clones                                                                      Plaque-                                  Library.sup.1                                                                         % Recomb..sup.2                                                                         Antibody.sup.3                                                                           # Screened                                                                            Purified                                 ______________________________________                                        PNF/RNA 85        PNF        5.5 × 10.sup.5                                                                  4                                        PNF/RNA 90        PNF          8 × 10.sup.4                                                                  7                                        TOTALS:                              11                                       ______________________________________                                         .sup.1 cDNA library constructed from the indicated human source.              .sup.2 Percent recombinant clones in the indicated λgt11 library a     determined by blue/white plaque assay and confirmed by PCR amplification      of randomly selected clones.                                                  .sup.3 Antisera source used for the immunoscreening of each indicated         library.                                                                 

One of the clones isolated by the above screen (PNF 2161 clone 470-20-1,SEQ ID NO:3; β-galactosidase in-frame fusion translated sequence, SEQ IDNO:4), was used to generate extension clones, as described in Example 6.Clone 470-20-1 nucleic acid sequence is presented as SEQ ID NO:3(protein sequence SEQ ID NO:4). The isolated nucleic acid sequencewithout the SISPA cloning linkers is presented as SEQ ID NO:19 (proteinSEQ ID NO:20).

EXAMPLE 4 Characterization of the Immunoreactive 470-20-1 Clone

A. Southern Blot Analysis of Immunoreactive Clones.

The inserts of immunoreactive clones were screened for their ability tohybridize to the following control DNA sources: normal human peripheralblood lymphocyte (purchased from Stanford University Blood Bank,Stanford, Calif.) DNA, and Escherichia coli KM392 genomic DNA (Ausubel,et al.; Maniatis, et al.; Sambrook, et al.). Ten micrograms of humanlymphocyte DNA and 2 micrograms of E. coli genomic DNA-were digestedwith EcoRI and HindIII. The restriction digestion products wereelectrophoretically fractionated on an agarose gel (Ausubel, et al.) andtransferred to nylon or nitrocellulose membranes (Schleicher andSchuell, Keene, N.H.) as per the manufacturer's instructions.

Probes from the immunoreactive clones were prepared as follows. Eachclone was amplified using primers corresponding to lambda gt11 sequencesthat flank the EcoRI cloning site of the gt11 vector. Amplification wascarried out by polymerase chain reactions utilizing each immunoreactiveclone as template. The resulting amplification products were digestedwith EcoRI, the amplified fragments gel purified and eluted from the gel(Ausubel, et al.). The resulting amplified fragments, derived from theimmunoreactive clones, were then random prime labelled using acommercially available kit (BMB) employing ³² p-dNTPs.

The random primed probes were then hybridized to the above-preparednylon membrane to test for hybridization of the insert sequences to thecontrol DNAs. The 470-20-1 insert did not hybridize with any of thecontrol DNAs.

As positive hybridization controls, a probe derivative from a humanC-kappa gene fragment (Hieter) was used as single gene copy control forhuman DNA and a E. coli polymerase gene fragment was similarly used forE. coli DNA.

B. Genomic PCR.

PCR detection was developed first to verify exogenicity with respect toseveral genomic DNAs which could have been inadvertently cloned duringlibrary construction, then to test for the presence of the clonedsequence in the cloning source and related specimen materials. Severaldifferent types of specimens, including SISPA-amplified nucleic acidsand nucleic acids extracted from the primary source, and nucleic acidsextracted from related source materials (e.g., from animal passagestudies), were tested.

The term "genomic PCR" refers to testing for the presence of specificsequences in genomic DNA from relevant organisms. For example, a genomicPCR for a Mystax-derived clone would include genomic DNAs as follows:

1. human DNA (1 μg/rxn.)

2. Mystax DNA (0.1-1 μg/rxn.)

3. E. coli (10-100 ng/rxn.)

4. yeast (10-100 ng/rxn.)

Human and Mystax DNAs are tested, as the immediate and ultimate sourcefor the agent. E. coli genomic DNA, as a frequent contaminant ofcommercial enzyme preparations, is tested. Yeast is also tested, as aubiquitous organism, whose DNA can contaminate reagents and thus, becloned.

In addition, a negative control (i.e., buffer or water only), andpositive controls to include approximately 10⁵ c/rxn., are alsoamplified.

Amplification conditions vary, as may be determined for individualsequences, but follow closely the following standard PCR protocol: PCRwas performed in reactions containing 10 mM Tris, pH 8.3, 50 mM KCl,1.75 mM MgCl₂, 1.0 μM each primer, 200 μM each DATP, dCTP, and dGTP, and300 μM dUTP, 2.5 units Taq DNA polymerase, and 0.2 unitsuracil-N-glycosylase per 100 ul reaction. Cycling was for at least 1minute at 940° C., followed by 30 to 40 repetitions of denaturation(92°-94° C. for 15 seconds), annealing (55°-56° C. for 30 seconds), andextension (720° C. for 30 seconds). PCR reagents were assembled, andamplification reactions were constituted, in a specially-designatedlaboratory maintained free of amplified DNA.

As a further barrier to contamination by amplified sequences and thuscompromise of the test by "false positives," the PCR was performed withdUTP replacing TTP, in order to render the amplified sequencesbiochemically distinguishable from native DNA. To enzymatically renderunamplifiable any contaminating PCR product, the enzymeuracil-N-glycosylase was included in all genomic PCR reactions. Uponconclusion of thermal cycling, the reactions were held at 72° C. toprevent renaturation of uracil-N-glycosylase and possible degradation ofamplified U-containing sequences.

A "HOT START PCR" was performed, using standard techniques ("AMPLIWAX",Perkin-Elmer Biotechnology; alternatively, manual techniques were used),in order to make the above general protocol more robust foramplification of diverse sequences, which ideally require differentamplification conditions for maximal sensitivity and specificity.

Detection of amplified DNA was performed by hybridization to specificoligonucleotide probes located internal to the two PCR primer sequencesand having no or minimal overlap with the primers. In some cases, directvisualization of electrophoresed PCR products was performed, usingethidium bromide fluorescence, but probe hybridization was in each casealso performed, to help ensure discrimination between specific andnon-specific amplification products. Hybridization to radiolabelledprobes in solution was followed by electrophoresis in 8-15%polyacrylamide gels (as appropriate to the size of the amplifiedsequence) and autoradiography.

Clone 470-20-1 was tested by genomic PCR, against human, E. Coli, andyeast DNAs. No specific sequence was detected in negative controlreactions, nor in any genomic DNA which was tested, and 10⁵ copies ofDNA/reaction resulted in a readily-detectable signal. This sensitivity(i.e., 10⁵ /reaction) is adequate for detection of single-copy humansequences in reactions containing 1 ug total DNA, representing the DNAfrom approximately 1.5×10⁵ cells.

C. Direct Serum PCR

Serum or other cloning source or related source materials were directlytested by PCR using primers from selected cloned sequences. In theseexperiments, HGV viral particles were directly precipitated from serawith polyethylene glycol (PEG), or, in the case of PNF and certain othersera, were pelleted by ultracentrifugation. For purification of RNA, thepelleted materials were dissolved in guanidinium thiocyanate andextracted by the acid guanidinium phenol technique (Chomczynski, etal.).

Alternatively, a modification of this method afforded through andimplemented by the use of commercially available reagents, e.g.,"TRIREAGENT" (Molecular Research Center, Cincinnati, Ohio) or "TRIZOL"(Life Technologies, Gaithersburg, Md.), and associated protocols wasused to isolate RNA. In addition, RNA suitable for PCR analysis wasisolated directly from serum or other fluids containing virus, withoutprior concentration or pelleting of virus particles, through the use of"PURESCRIPT" reagents and protocols (Gentra Systems, Minneapolis,Minn.).

Isolated DNA was used directly as a template for the PCR. RNA wasreverse transcribed using reverse transcriptase (Gibco/BRL), and thecDNA product was then used as a template for subsequent PCRamplification.

In the case of 470-20-1, nucleic acid from the equivalent of 20-50 ul ofPNF serum was used as the input template into each RT-PCR or PCRreaction. Primers were designed based on the 470-20-1 sequence, asfollows: 470-20-1-77F (SEQ ID NO:9) and 470-20-1-211R (SEQ ID NO:10).Reverse performed using MMLV-RT (Gibco/BRL) and random hexamers(Promega) by incubation at room temperature for approximately 10minutes, 42° C. for 15 minutes, and 99° C. for 5 minutes, with rapidcooling to 4° C. The synthesized cDNA was amplified directly, withoutpurification, by PCR, in reactions containing 1.75 mM MgCl₂, 0.2-1 μMeach primer, 200 uM each DATP, dCTP, dGTP, and dTTP, and 2.5-5.0 unitsTaq DNA polymerase ("AMPLITAQ", Perkin-Elmer) per 100 ul reaction.Cycling was for at least one minute at 94° C., followed by 40-45repetitions of denaturation (94° C. for 15 seconds for 10 cycles; 92° C.or 94° C. for 15 seconds for the succeeding cycles), annealing (55° C.for 30 seconds), and extension (72° C. for 30 seconds), in the "GENEAMPSYSTEM 9600" thermal cycler (Perkin-Elmer) or comparable cyclingconditions in other thermal cyclers (Perkin-Elmer; MJ Research,Watertown, Mass.).

Positive controls consisted of (i) previously amplified PCR productwhose concentration was estimated using the Hoechst 33258 fluroescenceassay, (ii) purified plasmid DNA containing the DNA sequence ofinterest, or (iii) purified RNA transcripts derived from plasmid clonesin which the DNA sequence of interest is disposed under thetranscriptional control of phage RNA promoters such as T7, T3, or SP6and RNA prepared through the use of commercially available in vitrotranscription kits. In addition, an aliquot of positive control DNAcorresponding to approximately 10-100 copies/rxn. can be spiked intoreactions containing nucleic acids extracted from the cloning sourcespecimen, as a control for the presence of inhibitors of DNAamplification reactions. Each separate extract was tested with at leastone positive control.

Specific products were detected by hybridization to a specificoligonucleotide probe 470-20-1-152F (SEQ ID NO:16), for confirmation ofspecificity. Hybridization of 10 ul of PCR product was performed insolution in 20 ul reactions containing approximately 1×10⁶ cpm of ³²P-labelled 470-20-1-152F. Specific hybrids were detected followingelectrophoretic separation from unhybridized oligo in polyacrylamidegels, and autoradiography.

In addition to PNF, extracted nucleic acids from normal serum was alsoreverse transcribed and amplified, using the "serum PCR" protocolsequence. No signal was detected in normal human serum. The specificsignal in PNF serum was reproducibly detected in multiple extracts, withthe 470-20-1 specific primers.

D. AMPLIFICATION FROM SISPA UNCLONED NUCLEIC ACIDS

SISPA (Sequence-Independent Single Primer Amplification) amplified CDNAwas used as templates (Example 1). Sequence-specific primers designedfrom selected cloned sequences were used to amplify DNA fragments ofinterest from the templates. Typically, the templates were theSISPA-amplified samples used in the cloning manipulations. For example,amplification primers 470-20-1-77F (SEQ ID NO:9) and 470-20-1-211R (SEQID NO:10) were selected from the clone 470-20-1 sequence (SEQ ID NO:3).These primers were used in amplification reactions with theSISPA-amplified PNF2161 cDNA as a template.

The identity of the amplified DNA fragments were confirmed by (i)hybridization with the specific oligonucleotide probe 470-20-1-152F (SEQID NO:16), designed based on the 470-20-1 sequence (SEQ ID NC:3) and/or(ii) size. The probe used for DNA blot detection was labelled withdigoxygenin using terminal transferase according to the manufacturer'srecommendations (BMB). Hybridization to the amplified DNA was thenperformed using either Southern blot or liquid hybridization (Kumar, etal., 1989) analyses.

Positive control DNA used in the amplification reactions was previouslyamplified PCR product whose concentration was estimated by the Hoechst33258 fluorescence assay, or, alternatively, purified plasmid DNAcontaining the cloned inserts of interest.

The 470-20-1 specific signal was detected in cDNA amplified by PCR fromSISPA-amplified PNF2161. Negative control reactions were nonreactive,and positive control DNA templates were detected.

E. Amplification from Liver RNA Samples.

RNA was prepared from liver biopsy material following the methods ofCathal, et al., wherein tissue was extracted in 5M guanidine thiocyanatefollowed by direct precipitation of RNA by 4M LiCl. After washing of theRNA pellet with 2M LiCl, residual contaminating protein was removed byextraction with phenol:chloroform and the RNA recovered by ethanolprecipitation.

The 470-20-1 specific primers were also used in amplification reactionswith the following RNA sources as substrate: normal mystax liver RNA,normal tamarin (Sanguinus labiatus) liver RNA, and MY131 liver RNA.MY131 is a mystax that was inoculated intravenously with 1 ml of PNF2161 plasma. There were obvious elevations of a liver enzyme (SCID) andhistological evidence of an apparent viral infection. The histologicalcorrelation was most obvious in the liver of MY131, whose liver wasobtained at or near the peak of SCID activity. Mystax 131 liver RNA didnot give amplified products with the non-coding primers (SEQ ID NO:7 andSEQ ID NO:8) of HCV.

The amplification reactions were carried out in duplicate for twoexperiments. The results of these amplification reactions are presentedin Table 2.

                  TABLE 2                                                         ______________________________________                                        PCR with 470-20-1 Primers                                                                    Exp. 1        Exp. 2                                                          A   B         A     B                                          ______________________________________                                        Normal My liver RNA                                                                            -     -         -   -                                        Normal tamarin liver RNA                                                                       -     -         -   -                                        My131 liver RNA  +     +         +   +                                        PNF 2161         ++    ++        ++  ++                                       ______________________________________                                    

These results demonstrate the 470-20-1 sequences are present in theparent serum sample (PNF 2161) and in a liver RNA sample from a passageanimal of the PNF 2161 sample (MY131). However, both control RNAs werenegative for the presence of 470-20-1 sequences.

F. Screening of a Serum Panel for HGV Sequences by Polymerase ChainReaction Using RNA Templates.

1. HIGH-ALT DONORS

The disease association between HGV and liver disease was assessed bypolymerase chain reaction screening, using HGV specific primers, of serafrom is hepatitis patients and from blood donors with abnormal liverfunction. The latter consisted of serum from blood donations with serumALT levels greater than 45 International Units per ml.

A serum panel consisting of 152 total sera was selected. The followingsera were selected for the serum panel: 104 high-ALT sera from screenedblood donations at the Stanford University Blood Bank (SUBB); 34N-(ABCDE) hepatitis sera from northern California, Egypt, and Peru; and14 sera from other donors suspected of having liver disease and/orhepatitis virus infection. The negative controls for the panel were asfollows: 9 highly-screened blood donors (SUBB) notable for the absenceof risk factors for viral infections ("supernormal" sera, e.g.,O-negative, Rh-negative; negative for HIV, known hepatitis agents, andCMV; whose multiple previous blood donations had been transfused withoutcausing disease); and 2 random blood donors. These sera were assayed forthe presence of HGV specific sequences by RT-PCR using the 470-20-1primers 77F (SEQ ID NO:9) and 211R (SEQ ID NO:10).

RNA extraction and RT-PCR were performed essentially as described inExample 4C, except that the primer 470-20-1-211R was 5'-biotinylated tofacilitate rapid screening of amplified products by a method involvinghybridization in solution, followed by affinity capture of hybridizedprobe using streptavidin-coated paramagnetic beads. Methods for theanalysis of nucleic acids by hybridization to specific labelled probeswith capture of the hybridized sequences through affinity interactionsare well known in the art of nucleic acid analysis.

Depending on the amount of serum available for testing, RNA from 30 to50 μl of serum was used per RT/PCR reaction. Each serum was tested induplicate, with positive controls corresponding to 10, 100, or 1000copies of RNA transcript per reaction and with appropriate negative(buffer) controls. No negative controls were reactive, and at least 10copies per reaction were detectable in each PCR run. Indeterminateresults were defined as specific hybridizing signal being present inonly one of two duplicate reactions.

Efficient, highly sensitive analysis of the products from theamplification analysis of this serum panel was performed using aninstrument specifically designed for affinity-based hybrid capture usingelectrochemiluminscent oligonucleotide probes (QPCR System 5000™,Perkin-Elmer). Assays utilizing the QPCR 5000™ have been described(DiCesare, et al; Wages, et al).

The products of each reaction were assayed by hybridization to probe470-20-1-152F (5'-end-labelled with an electrochemiluminescent rutheniumchelate), and measurement using the "QPCR 5000." Based on a cutoff ofthe sum of the mean and three times the standard deviation of negativecontrols in a given amplification run, a total of 34 possible positiveswere selected for confirmatory testing.

The 34 samples were analyzed by solution hybridization andelectrophoresis (Example 4C). Out of these 34 samples, 6 sera (i.e.,6/152) were shown to have specific hybridizing sequences in duplicatereactions. Of these six samples, three were strongly reactive bycomparison with positive controls: one High-ALT serum from SUBB, and twoN-(ABCDE) sera from Egypt.

A second blood sample was obtained from the highly positive SUBB serumdonor one year after the initial sample was taken. The second serumsample was confirmed to be HGV positive by the PCR methods describedabove. This result confirms persistant infection by HGV in a human. Theserum was designated "JC." Further, the serum donor was HCV negative(determined by seroreactivity tests and PCR) and antibody negative forHAV and HBV.

In addition, a third N-(ABCDE) serum from Egypt, a northern Californiablood donor with N-(ABCDE) hepatitis, and a N-(ABCDE) hepatitis serum,were also shown to be weakly positive by this method. Two other seragave indeterminate results, defined as the presence of specificsequences in one of two amplification reactions.

Subsequent PCR analysis of replicate serum aliquots from theseHGV-positive and indeterminate sera resulted in HGV-positive results in6 of 8 sera tested and indeterminate results in the remaining 2 sera.

A second primer set was used for the confirmation of HGV positivesamples. This primer set (GV57-4512MF, SEQ ID NO:121, and GV57-4657MR,SEQ ID NO:122) for diagnostic amplification, was selected from aconserved region of HGV derived from the putative NS5 coding region. Anapproximately 2.2 kb fragment was amplified from each of 5 separate HGVisolates. The primers used for the amplification reactions were470EXT4-2189R (SEQ ID NO:119) and 470EXT4-29F (SEQ ID NO:120). Theamplified DNA fragments were sequenced and the sequences aligned. Highlyconserved regions were identified from the alignment and optimal primersequences were designed incorporating mixed base synthesis at thosepositions that remained divergent throughout the five sequences. Theresulting NS5 primers were as follows: GV57-4512MF, SEQ ID NO:121, andGV57-4657MR, SEQ ID NO:122. These primers were used to amplify adiagnostic fragment of 165 bp from test samples.

An internal probe sequence, GV22dc-89MF (SEQ ID NO:123) was derived fromanother highly conserved region for detection of the specificallyamplified product. The probe is also of sufficient length to allow fordetection of minimally divergent HGV sequences under lowered stringencyconditions.

Analysis of specimens for the presence of the diagnostic NS5 sequencefollowed the same conditions for sample preparation, amplification, andliquid hybridization as described for the 470-20-1 primers (Example 4C).The concordance of results for sera samples analyzed by PCR using boththe 470-20-1 and NS5 primer pairs are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                470-20-1 Primer Pair                                                                 +      -      Indeterminant                                    ______________________________________                                        NS5-Region                                                                              +          71       0    1                                          Primer    -          6        13   2                                          Pair      Indeterminant                                                                            2        1    0                                          (GV57)                                                                        ______________________________________                                    

Further PCR analyses of additional aliquots obtained from the 8 seraidentified above as being HGV-positive were carried out using the470-20-1 primer set (SEQ ID NO:9 and SEQ ID NO:10) and the NS5 primerset. In these assays, the HGV PCR analyses gave consistently positiveresults in 5 of the 8 sera. These results are presented in Table 4.

In contrast, none of the two random donors or nine highly-screened"supernormal" sera was positive in either set of PCR analysis.

These results reinforce the disease association between HGV and liverdisease.

                  TABLE 4                                                         ______________________________________                                                          Number  Number                                              Specimen Group    Tested  Positive                                            ______________________________________                                        High-ALT Donor    104     1                                                   Non-ABCDE, other   48     4                                                   Normal Donor       2      0                                                   "Supernormal"      9      0                                                   Totals            163     5                                                   ______________________________________                                    

Further testing of sera from High-ALT donors has yielded the followingresults. A total of 495 sera have been tested, in addition to theinitial panel of 104 sera described above. Of these 495 specimens, 6were identified as HGV positive using the primer pair 470-20-1-77F (SEQID NO:9) and 470-20-1-211R (SEQ ID NO:10). These six sera have thefollowing HCV profiles: R25342, HCV negative; R17749, HCV positive;J53171, HCV positive, HBV positive; J54406, HCV negative; R08074, HCVnegative; and X31049, HCV negative. Positive scores are based onrepeated reactivity in at least 2 separate reactions. R25342 was testedand confirmed positive by PCR using the NS5 primer pair. Accordingly, adetection rate of approximately 1.2% has been observed (7 of 599tested).

Freshly-obtained plasma samples from blood donors with elevated ALT werealso obtained from SUBB, the Peninsula Blood Bank (Burlingame, Calif.),and the New York Blood Center (New York, N.Y.), for testing for HGV RNAby PCR (470-20-1 primer pair). Of 214 total donations which were tested,a total of 5 (approximately 2.3%) were HGV RNA positive. These five serahave the following HCV profiles: T55806, HCV positive; T55875, HCVnegative; T56633, HCV negative; R38730, HCV negative; and 3831781, HCVnegative. Subsequent donations from two of these donors, T55806 andT55875, were also HGV RNA positive. T55806, T55875 and T56633 weretested and confirmed positive by PCR using the NS5 primer pair.

2. SCREENING OF ACCEPTED BLOOD DONORS

To assess the prevalence of HGV in the normal blood donor population,serum was collected from screened blood donors for transfusion at SUBB.A total of 968 specimens, representing 769 unique donors, was tested forHGV RNA. The samples were screened by PCR using the 470-20-1 primerpair.

A total of 16 sera were identified as having detectable HGV RNA. Ofthese, 6 represent duplicates from 3 donors, such that a total of 13unique donors of 769 tested were HGV positive by RNA PCR. All positivesamples were tested and confirmed positive by PCR using the NS5 primerpair. These donors were characterized by normal ALT levels, as well asotherwise normal serology. Accordingly, approximately 1.7% of the seratested in the normal blood donor population are HGV positive. Therefore,the presence of HGV was detected in both accepted and rejected blooddonors.

3. SPECIMENS FROM VARIOUS GEOGRAPHIC LOCALES.

The presence of HGV infection in populations of hepatitis patients fromgeographically widespread sources was assessed by PCR. The PCR reactionswere carried out essentially as described in Example 4C using the470-20-1 PCR primer pairs. Serum samples from Egypt, Greece, Australia(see Example 4F-4), Peru, England, Italy, Germany, South Korea, UnitedStates and Japan were tested. HGV RNA was detectable in subsets of allpopulations tested.

4. POST-TRANSFUSION ASSOCIATED HGV INFECTION AND PARENTERALTRANSMISSION.

HGV RNA was detected in several post-transfusion hepatitis cases (thoseof Japanese and European origin were included in Example 4F-3). For 4total cases, one from Japan, two from the U.S. and one from Australia,multiple time-points were assayed for the presence of HGV RNA. For 3 ofthese cases, (i) pre-transfusion samples were available to estabishprevious HGV status of the patient, and (ii) samples were available fromindividual blood donors to those three cases, to establish donor HGVstatus.

The first case was a Japanese patient transfused on Dec. 2, 1980.Following the transfusion the patient developed Non-B Non-C hepatitis. Atotal of 5 sera from this patient were tested for HGV RNA by PCR usingthe 470-20-1 primer pair. HGV RNA was detectable from about 2 weeks toabout 8 months following transfusion. A sample taken greater than 1 yearpost-transfusion was indeterminate (i.e., positive in one duplicatereaction only). No pre-transfusion sample was available for testing.

Cases BIZ and STO (Tables 5 and 6, respectively) were from aprospectively-followed heart surgery study (Alter, et al., 1989)conducted at the NIH. For each of these patients, pre-transfusion serawere available and were determined to be negative for HGV RNA by PCRusing the 470-20-1 primer pair. BIZ tested positive for HGV RNA from dayone post-transfusion to week 198 post-transfusion. Of 9 total blooddonors to BIZ, 2 out of 8 tested were found to be HGV positive. STOtested positive for HGV RNA from week 5 post-transfusion through week 92post-transfusion.

                  TABLE 5                                                         ______________________________________                                        Transfusion-Associated Transmission                                           of HGV: Case BIZ                                                              Draw                    ALT in  470 PCR                                       Date        Time        IU/L    Result                                        ______________________________________                                        10/30/78     -4 days    23      -                                             11/01/78     -1 day     31      -                                             11/03/78     +1 day     29      +                                             11/17/78     2 weeks    51      +                                             03/22/79     +20 weeks  135     +                                             06/28/79     +34 weeks  133     +                                             04/06/81    +127 weeks  141     +                                             08/20/82    +198 weeks  39      +                                             ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Transfusion-Associated Transmission                                           of HGV: Case STO                                                              Draw                    ALT in  470 PCR                                       Date        Time        IU/L    Result                                        ______________________________________                                        06/15/83     -1 day     23      -                                             07/18/83     +5 weeks   80      +                                             10/31/83    +20 weeks   75      +                                             12/31/83    +28 weeks   30      +                                             01/02/85    +81 weeks   90      -                                             03/20/85    +92 weeks   23      +                                             ______________________________________                                    

The fourth case, also prospectively-defined, was a cardiac surgerypatient who participated in a post-transfusion hepatitis study conductedin Sydney, Australia. The patient (PA-124), having no other identifiablerisk factors, received 14 units of blood during surgery (4 units packedred cells, 10 units of platelets). Of these 14 units one was HGVpositive; the other 13 were HGV negative. HBV and HCV serologies of the14 blood donors were negative with the exception of a reactive HCV EIA(first generation test). No other HCV test confirmed the positivefinding.

In patient PA-124 (Table 7), serum ALT was elevated beginning with asample taken two weeks post-operation, and was observed to be at least10 times the pre-operation level for a period of 14 weeks. PCR resultsfor HCV performed on pre-transfusion, 4 week, and 8 week sera fromPA-124, were all negative. Serum from this patient was tested for HGVRNA using the 470-20-1 PCR primers. A pre-transfusion sample wasnegative for HGV RNA. Positive results were demonstrated followingtransfusion, coinciding with and succeeding the ALT elevation. Thepresence of HGV RNA was detected out to one year post-transfusion. Thesedata support the conclusion that HGV may be parenterally transmitted.

                  TABLE 7                                                         ______________________________________                                        Transfusion-Associated Transmission                                           of HGV: Case PA-124                                                           Weeks            ALT in  470 PCR                                              Post-Operation   IU/L    Result                                               ______________________________________                                        pre-transfusion   7      -                                                     2               74      +                                                     4               86      +                                                     8               135     +                                                    12               179     +                                                    14               78      +                                                    18                9      +                                                    24                6      +                                                    36               11      +                                                    52               11      +                                                    64               23      -                                                    84               10      -                                                    ______________________________________                                    

In addition to prospectively-defined post-transfusion transmissioncases, additional cases of HGV infection were identified in risk groupsdefined by multiple transfusions and intravenous drug use (IVDU) (Table8).

                  TABLE 8                                                         ______________________________________                                        HGV RT-PCR Testing of Coded Sera:                                             Selected Hepatitis and Parenteral Risk Groups                                 Group           Number Tested                                                                            Number Positive                                    ______________________________________                                        Autoimmune Hepatitis                                                                          10         0                                                  Primary Biliary Cirrhosis                                                                     20         0                                                  Suspected Acute NonA-E                                                                        24         2                                                  Hepatitis                                                                     Chronic Hepatitis (NonA-C                                                                     34         3                                                  (confirmed by liver                                                           biopsy)                                                                       Heptaocellular Carcinoma                                                                      20         2                                                  Chronic HBV     20         2                                                  Chronic HCV     50         6                                                  Hemophilia      49         9                                                  IVDU            54         15                                                 Multiply Transfused Anemia                                                                    100        19                                                 ______________________________________                                    

Among 100 multiply-transfused sickle cell anemia and thalassemiapatients, 19 (19%) were found to have detectable serum HGV RNA.Similarly, 9 of 49 hemophilia patients (18%) were HGV positive with470-20-1 and NS5 primers. Significantly, 15 of 54 (28%) IVDU were foundto be PCR positive for HGV RNA. Infection rates in these parenteral riskgroups (18-28%) appear to be higher than rates in blood donors withelevated ALT (1-2%). These results reinforce the significance of theparenteral route for HGV transmission.

5. PCR SCREENING OF SELECTED HEPATITIS DISEASE GROUPS

Sera from patients with acute and chronic hepatitis, hepatocellularcarcinoma, HBV infection or HCV infection were tested for the presenceof HGV using polymerase chain reaction (data presented in Table 8). Ineach of sets of specimens from patients with liver disease, HGV positivespecimens were demonstrated (with the exception of specimens frompatients with autoimmune hepatitis and primary biliary cirrhosis, bothconditions not thought to be exclusively associated with an infectiousagent).

As shown in the collections of sera from post-transfusion hepatitispatients (Example 4F-4), HGV infection is established during acutehepatitis, but circulating viral RNA continues to be detected duringchronic infection for periods of time measured in months to years.

Approximately 10-20% co-infection rates were observed in patients withHBV and HCV infection. HGV infection is thus shown to be associated withhepatitis with or without co-infection with other hepatitis viruses.Co-infection may reflect similar risk factors and routes of transmissionfor these hepatitis viruses. As noted above, there is a higherprevalence of HGV in parenteral risk groups, such as hemophiliacs,IVDU's, and multiply transfused anemia patients (compared with otherhepatitis risk groups).

6. PERSISTENT INFECTION BY HGV IN HUMANS

Post-transfusion hepatitis cases BIZ, STO, and PA-124 were show to havePCR-detectable viral RNA up to 3.8, 1.8, and 1.0 years, respectively,following transfusion and acute infection. Additional serum samples wereobtained from donor JC (Example 4F-1), one year and 1.5 years followingthe initial positive sample. These follow-up serum samples were also HGVpositive. Additional sera from other high-ALT donors (T55806, T55875,R25342), obtained several months following the serum sample in which HGVinfection was originally detected, were also positive. Similarly, whenHGV infection was established in an experimental primate (CH1356,Example 4H), HGV RNA was detected over 1.5 years following innoculation.These data establish persistent HGV viremia in humans and experimentalprimates.

G. Amplification of Long Fragments from Patient RNA for Sequencing.

PCR primers were designed to amplify several informative regions of theHGV genome in order to obtain sequence information on varied HGVisolates. The primers 470EXT4-2189R (SEQ ID NO:119) and 470EXT4-29F (SEQID NO:120) were designed to amplify a 2.2 kb fragment that contained theoriginal 470-20-1 sequence. RNA from samples was reverse-transcribedusing "SUPERSCRIPT II" reverse transcriptase (Gibco/BRL, Gaithersburg,Md.). The resulting cDNA was amplified using reagents for efficientlong-range PCR ("XL PCR BUFFERS" and "rTth-XL", Perkin Elmer/AppliedBiosystems Div., Foster City, Calif.).

The amplification reaction was considered to be positive if a band ofthe correct size on agarose gel electrophoresis was detected. The samplewas confirmed as positive by preliminary DNA sequencing of theamplification product. The following sera samples tested positive forHGV RNA by this amplification method: PNF2161; R10291 (JC); andspecimens from each of the North American, Egyptian, and Japanesegroups. However, no positive samples were detected from the Peruviansera.

Successful amplification from a variety of HGV-positive specimensprovides confirmation of the results obtained by PCR amplification usingthe 470-20-1 primer pair discussed above. Failure to obtainamplification, however, may reflect poor RNA quality or low copy numberor local sequence differences among isolates such that the selectedprimer sets would not function universally.

In order to obtain sequence information from the putative5'-untranslated region of the HGV genome, primers were designed toamplify fragments from the 5'-untranslated region (based on the HGV PNF2161-variant). The two fragments were defined by the following primersets: FV94-22F (SEQ ID NO:124) and FV94-724R (SEQ ID NO:125), yielding a728 base pair fragment; and FV94-94F (SEQ ID NO:126) and FV94-912R (SEQID NO:127), yielding an 847 base pair fragment.

The conditions just described to promote efficient long-range PCR wereused. Products were obtained from most of the samples tested, providingadditional confirmation of the presence of HGV RNA in the samples.

H. Infectivity of HGV in Primates.

Two chimpanzees (designated CH1323 and CH1356), six cynomolgus monkeys(CY143, CY8904, CY8908, CY8912, CY8917, and CH8918), and six Mystax(MY29, MY131, MY98, MY187, MY229, MY254) subjects were inoculated withPNF 2161. Pre-inoculation and post-inoculation sera were monitored forALT and for the presence of HGV RNA sequences (as determined by PCRscreening--described above).

One cynomologous monkey (CY8904) showed a positive RNA PCR result (39days post-inoculation) and one indeterminant result from a total of 17seperate blood draws. In one chimpanzee, designated CH1356, wassustained viremia observed by RT-PCR. As shown in Table 9, nosignificant ALT elevation was observed, and circulating virus wasdetected only at time points considerably after inoculation. Viremia wasobserved at and following 118 days post-inoculation. Suggestivereactivity was also observed in the first post-inoculation time-point (8days), which may indicate residual inoculum.

                  TABLE 9                                                         ______________________________________                                        ALT and PCR Results from CH1356 Following                                     Inoculation with PNF 2161                                                     Days Post-                                                                    Inoculation     ALT*     HGV PCR                                              ______________________________________                                         0              59       -                                                     8              65       ±                                                 15              85       -                                                    22              89       -                                                    29              89       -                                                    36              86       -                                                    39              31       -                                                    47              74       -                                                    54              40       -                                                    61              57       -                                                    84              65       ±                                                 89              63       +                                                    98              64       -                                                    118             84       +                                                    125             73       +                                                    134             74       +                                                    159             80       +                                                    610             (ALT not +                                                                    available)                                                    ______________________________________                                         *average ALT baseline before inoculation was 50.                         

The data presented above indicate that HGV infection was persistent upto 1.7 years in an experimental primate.

I. Characterization of the Viral Genome.

The isolation of 470-20-1 from a cDNA library (Example 1) suggests thatthe viral genome detected in PNF 2161 is RNA. Further experiments toconfirm the identity of the HGV viral genome as RNA include thefollowing.

Selective degradation of either RNA or DNA (e.g., by DNase-free RNase orRNase-free DNase) in the original cloning source followed byamplification with HGV specific primers and detection of theamplification products serves to distinguish RNA from DNA templates.

An alternative method makes use of amplification reactions (nucleicacids from the original cloning source as template and HGV specificprimers) that employ (i) a DNA-dependent DNA polymerase, in the absenceof any RNA-dependent DNA polymerase (i.e., reverse transcripase) in thereactions, and (ii) a DNA-dependent DNA polymerase and an RNA-dependentDNA polymerase in the reactions. In this method, if the HGV genome isDNA or has a DNA intermediate, then amplified product is detected inboth types of amplification reactions. If the HGV genome is only RNA,the amplified product is detected in only the reversetranscriptase-containing reactions.

Total nucleic acid (i.e., DNA or RNA) was extracted from PNF 2161, usingproteinase K and SDS followed by phenol extraction, as described inExample 4C. The purified nucleic acid was then amplified usingpolymerase chain reaction (PCR) where either (i) the PCR was preceded bya reverse transcription step, or (ii) the reverse transcription step wasomitted. Amplification was reproducibly obtained only when the PCRreactions were preceded by reverse transcription. As a control, DNAtemplates were successfully amplified in separate reactions. Theseresults demonstrate that the nature of the HGV viral genome is RNA.

The strand of the cloned, double-stranded DNA sequence that wasoriginally present in PNF 2161 may be deduced by various means,including the following. Northern or dot blotting of the unamplifiedgenomic RNA from an infected source serum can be performed, followed byhybridization of duplicate blots to probes corresponding to each strandof the cloned sequence. Alternatively, single-stranded cDNA probesisolated from M13 vectors (Messing), or multiple strand-specificoligonucleotide probes are used for added sensitivity. If the sourceserum contains single-stranded RNA, only one probe (i.e., sequences fromone strand of the 470-20-1 clones) yield a signal, under appropriateconditions of hybridization stringency. If the source serum containsdouble-stranded RNA, both strand-probes will yeild a signal.

The polymerase chain reaction, prefaced by reverse transcription usingone or the other specific primer, represents a much more sensitivealternative to Northern blotting. Genomic RNA extracted from purifiedvirions present in PNF 2161 serum is used as the input template intoeach RT/PCR. Rather than cDNA synthesis with random hexamers, HGVsequence-specific primers were used. One cDNA synthesis reaction wasperformed with a primer complementary to one strand of the clonedsequence (e.g., 470-20-1-77F); a second cDNA synthesis reaction was alsoperformed using a primer derived from the opposite strand (e.g.,470-20-1-211R).

The resulting first strand cDNA was amplified in using two HGV specificprimers. Controls were included for successful amplification by PCR(e.g., DNA controls). RNA transcripts from each strand of the clonedsequence was also used, to control also for the reverse transcriptionefficiency obtained when using the specific primers which are described.

Specific products were detected by agarose gel electrophoresis withethidium bromide staining. DNA controls (i.e., double-stranded DNAcontrols for the PCR amplifcation) were successfully amplifiedregardless of the primer used for reverse transcription. Single-strandedRNA transcripts (i.e., controls for reverse transcription efficiency andstrand specificity) were amplified only when the opposite-strand primerwas used for cDNA synthesis.

The PNF-derived HGV polynucleotide gave rise to a specific amplifiedproduct only when the primer 470-20-1-211R was used for reversetranscription, thus indicating that the original HGV polynucleotidesequence present in the serum is complementary to 470-20-1-211R and islikely a single-strand RNA.

EXAMPLE 5 Sucrose Density Gradient Separation of PNF2161

A. Banding of PNF-2161 Agent.

A continuous gradient of 10-60% sucrose ("ULTRAPURE", Gibco/BRL) in TNE(50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM EDTA) was prepared using agradient maker from Hoefer Scientific (San Francisco, Calif.).Approximately 12.5 ml of the gradient was overlaid with 0.4 ml of PNFserum which had been stored at -70° C., rapidly thawed at 37° C., thendiluted in TNE.

The gradient was then centrifuged in the SW40 rotor (BeckmanInstruments) at 40,000 rpm (approximately 200,000×g at r_(av)) at 4° C.for approximately 18 hours. Fractions of volume approximately 0.6 mlwere collected from the bottom of the tube, and 0.5 ml was weigheddirectly into the ultracentrifuge tube, for calculation of density.

                  TABLE 10                                                        ______________________________________                                        Measured Densities of PNF Fractions                                           and Presence of 470-20-1                                                      Fraction     Density 470-20-1 Detected*                                       ______________________________________                                         1           1.274   -                                                         2           1.274   -                                                         3           1.266   -                                                         4           1.266   -                                                         5           1.260   -                                                         6           1.254   -                                                         7           1.248   +                                                         8           1.206   +                                                         9           1.146   +                                                        10           1.126   +++                                                      11           1.098   ++++                                                     12           1.068   +++                                                      13           1.050   +                                                        14           1.034   +                                                        15           1.036   +                                                        16           1.018   -                                                        17           1.008   +                                                        18           1.020   +                                                        ______________________________________                                         *"+" and "-" scores were initially based on 40cycle PCR. In order to          distinguish "+", "++", "+++", and "++++", fractions giving initial            positive scores (7-18) were amplified with 30 cycles of PCR.             

The putative viral particles were then pelleted by centrifugation at40,000 rpm in the Ti70.1 rotor (approximately 110,000×g) at 4° C. for 2hours, and RNA was extracted using the acid guanidinium phenol technique("TRI REAGENT", Molecular Research Center, Cincinnati, Ohio), andalcohol-precipitated using glycogen as a carrier to improve recovery.The purified nucleic acid was dissolved in an RNase-free buffercontaining 2 mM DTT and 1 U/μl recombinant RNasin.

Analysis of the gradient fractions by RNA PCR (Example 4C) showed adistinct peak in the 470-20-1 specific signal, localized in fractions ofdensity ranging from 1.126 to 1.068 g/ml (Table 10). The 470-20-1 signalwas thus shown, under these conditions, to form a discrete band,consistent with the expected behavior of a viral particle in a sucrosegradient.

B. Relative Viral Particle Densities.

PNF 2161 has been demonstrated to be co-infected with HCV (see above).In order to compare the properties of the 470-20-1 viral particle toother known hepatitis viral particles, the serum PNF 2161 and a sampleof purified Hepatitis A Virus were layered on a sucrose gradient (asdescribed above). Fractions (0.6 ml) were collected, pelleted and theRNA extracted. The isolated RNA from each fraction was subjected toamplification reactions (PCR) using HAV (SEQ ID NO:5; SEQ ID NO:6), HCV(SEQ ID NO:7; SEQ ID NO:8) and 470-20-1 (SEQ ID NO:9, SEQ ID NO:10)specific primers.

Product bands were identified by electrophoretic separation of theamplification reactions on agarose gels followed by ethidium bromidestaining. The results of this analysis are presented in Table 11.

                  TABLE 11                                                        ______________________________________                                        Average Density                                                                            HAV        HCV    470-20-1                                       ______________________________________                                        1.269        -          -      -                                              1.263        +          -      -                                              1.260        +          -      -                                              1.246        ++         -      -                                              1.238        ++         -      -                                              1.240        +          -      -                                              1.207        +          -      -                                              1.193        +          -      -                                              1.172        +          ±   -                                              1.150        +          ±   ±                                           1.134        +          +      ±                                           1.118        +          +      +                                              1.103        +          +      +                                              1.118        +          +      +                                              1.103        +          +      +                                              1.088        ±       +      +                                              1.084        -          +      +                                              1.080        -          +      +                                              1.070        -          +      +                                              1.057        -          +      +                                              1.035        -          ±   -                                              1.017        -          -      -                                              1.009        -          -      -                                              ______________________________________                                    

These results suggest that 470-20-1 particles are more similar to HCVparticles than to HAV.

Further, serum PNF 2161 and HAV particles were treated with chloroformbefore sucrose gradient centrifugation. The results of these experimentssuggest that 470-20-1 agent may be an enveloped virus since it has moresimilar properties to an enveloped Flaviviridae member (HCV) than anon-enveloped virus (HAV).

EXAMPLE 6 Generation of 470-20-1 Extension Clones

A. Anchor PCR.

RNA was extracted directly from PNF2161 serum as described in Example 1.The RNA was passed through a "CHROMA SPIN" 100 gel filtration column(Clontech) to remove small molecular weight impurities. cDNA wassynthesized using a BMB cDNA synthesis kit. After cDNA synthesis, thePNF cDNA was ligated to a 50 to 100 fold excess of KL-1/KL-2 SISPA orJML-A/JML-B linkers (SEQ ID NO:11/SEQ ID NO:12, and SEQ ID NO:17/SEQ IDNO:18, respectively) and amplified for 35 cycles using either the primerKL-1 or the primer JML-A.

The 470 extension clones were generated by anchored PCR of a 1 μlaliquot from a 10 μl ligation reaction containing EcoRI digested(dephosphorylated) lambda gt11 arms (1 μg) and EcoRI digested PNF cDNA(0.2 μg). PCR amplification (40 cycles) of the ligation reaction wascarried out using the lambda gt11 reverse primer (SEQ ID NO:13) incombination with either 470-20-77F (SEQ ID NO:9) or 470-20-1-211R (SEQID NO:10). All primer concentrations for PCR were 0.2 μM.

The amplification products (9 μl/100 μl) were separated on a 1.5%agarose gel, blotted to "NYTRAN" (Schleicher and Schuell, Keene, N.H.),and probed with a digoxygenin labelled oligonucleotide probe specificfor 470-20-1. The digoxygenin labeling was performed according to themanufacturer's recommendations using terminal transferase (BMB). Bandsthat hybridized were gel-purified, cloned into the "TA CLONING VECTORpCR II" (Invitrogen), and sequenced.

Numerous clones having both 5' and 3' extensions to 470-20-1 wereidentified. All sequences are based on a consensus sequence from thesequencing of at least two independent isolates. This Anchor PCRapproach was repeated in a similar manner to obtain further 5' and 3'extension sequences. These PCR amplification reactions were carried outusing the lambda gt11 reverse primer (SEQ ID NO:13) in combination withHGV specific primers derived from sequences obtained from previousextension clones. The substrate for these reactions was unpackaged PNF2161 2-cDNA source DNA.

Sequencing was carried out using "DYEDEOXY TERMINATOR CYCLE SEQUENCING"(a modification of the procedure of Sanger, et al.) on an AppliedBiosystems model 373A DNA sequencing system according to themanufacturer's recommendations (Applied Biosystems, Foster City,Calif.). Sequence data is presented in the Sequence Listing. Sequenceswere compared with "GENBANK", EMBL database and dbEST (National Libraryof Medicine) sequences at both nucleic acid and amino acid levels.Search programs FASTA, BLASTP, BLASTN and BLASTX (Altschul, et al.)indicated that these sequences were novel as both nucleic acid and aminoacid sequences.

Individual clones obtained using a selected primer pair were aligned toyield a consensus sequence. The series of consensus sequences used toconstruct the sequence for the HGV-PNF 2161 variant was as follows: 4E3,SEQ ID NO:26; 3E3, SEQ ID NO:27; 2E5, SEQ ID NO:28; 1E5, SEQ ID NO:29;4E5, SEQ ID NO:30; 3E5, SEQ ID NO:31; 2E3, SEQ ID NO:32; 1E3, SEQ IDNO:33; 4E5-20, SEQ ID NO:34; 5E3, SEQ ID NO:39; 6E3, SEQ ID NO:40; 7E3,SEQ ID NO:42; 5E5, SEQ ID NO:43; 6E5(44F), SEQ ID NO:44; 8E3, SEQ IDNO:98; 9E3, SEQ ID NO:109; 10E3, SEQ ID NO:110; 11E3, SEQ ID NO:116;12E3, SEQ ID NO:118; 5'-end, SEQ ID NO:175; and 3'-END, SEQ ID NO:167.

The individual consensus sequences were aligned, overlapping sequencesidentified and a consensus sequence for the HGV-PNF 2161 variant wasdetermined. This consensus sequence was compared with the sequencesobtained for four other HGV variants: JC (SEQ ID NO:182), BG34 (SEQ IDNO:176), T55806 (SEQ ID NO:178), and EB20-2 (SEQ ID NO:180).

The consensus sequence of the HGV-PNF 2161 variant consists of 9391 basepairs presented as SEQ ID NO:14. This sequence represents a continuousopen reading frame (SEQ ID NO:15). A Kyte-Doolittle hydrophobicity plotof the polyprotein is presented as FIG. 11.

The relationship between the original 470-20-1 clone and the sequencesobtained by extension is shown schematically in FIG. 1. As seen in thefigure, the DNA strand having opposite polarity to the protein codingsequence of 470-20-1 comprising a long continuous open reading frame.

The amino acid sequence of HGV was compared against the sequences of allviral sequence in the PIR database (IntelliGenetics, Inc., MountainView, Calif.) of protein sequences. The comparison was carried out usingthe "SSEARCH" program of the "FASTA" suite of programs version 1.7(Pearson, et al.). Regions of local sequence similarities were foundbetween the HGV sequences and two viruses in the Flaviviridae family ofviruses. The similarity alignments are presented in FIGS. 5A and 5B.

Present in these alignments are motifs for the RNA dependent RNApolymerase (RDRP) of these viruses. Conserved RDRP amino acid motifs areindicated in FIGS. 5A and 5B by stars and uppercase, bold letters(Koonin and Dolja). These alignments demonstrate that this portion ofthe HGV coding sequence correspond to RDRP. This alignment data combinedwith the data concerning the RNA genome of HGV supports the placement ofHGV as a member of the Flaviviridae family.

The global amino acid sequence identities of the HGV polyprotein (SEQ IDNO:15) with HOCV (Hog Cholera Virus) and HCV are 17.1% and 25.5%,respectively. Such levels of global sequence identity demonstrates thatHGV is a separate viral entity from both HOCV and HCV. To illustrate, intwo members of the Flaviviridae family of viruses BVDV (Bovine DiarrheaVirus) and HCV, 16.2% of the amino acids can be globally aligned withHGV.

Members within a genus generally show high homology when alignedglobally, for example, BVDV vs. HoCV show 71.2% identity. Variousmembers (variants) of the un-named genus of which HCV is a member arebetween 65% and 100% identical when globally aligned.

B. Race PCR: 5' End Cloning.

Clones representing the 5'-end of the HGV genome were obtained by amodified Anchor PCR approach that utilized RACE (Rapid Amplification ofcDNA Ends) technology. The RACE method was originally described byFrohman, et al., (1988) and Belyausky, et al., (1989). Briefly, the5'-end clones of HGV were obtained as follows.

First-strand cDNA synthesis was primed using random hexamers andsynthesis was carried out using either "SUPERSCRIPT II" or "rTth"reverse transcriptase (GIBCO/BRL). After first-strand synthesis, the RNAtemplate was degraded by base hydrolysis (NaOH). The cDNA sample wasneutralized by the addition of acetic acid and purified by absorption toa glass matrix support ("GENO-BIND," Clontech, Palo Alto, Calif.).Following purification, the cDNA was concentrated by ethanolprecipitation and washed twice with 80% ethanol.

The originally described RACE method was modified as follows. Asingle-stranded oligonucleotide anchor (SEQ ID NO:174) (Clontech) wasligated to the 3' end of the first-strand cDNA using T4 RNA ligase inthe presence of cobalt chloride. The oligonucleotide anchor was obtainedfrom the manufacturer with two modifications: (i) the 3'-end of theanchor was modified with an amino group which prevents concatamerformation, and (ii) the 5'-end contains a phosphate group which allowsligation to the first-strand cDNA.

After ligation of the anchor, the cDNA was used as a template for PCRamplification using several HGV-specific primers in combination with aprimer complementary to the anchor sequence (AP primer, SEQ ID NO:134).The resulting amplification products were separated by agarose gelelectrophoresis, transferred to filters and hybridized with a nested,HGV-specific oligonucleotide probe. Bands that hybridized to theHGV-probe were isolated, cloned into "pCR-II" (Invitrogen, San Diego,Calif.) and sequenced.

C. HGV 3' End Cloning.

Clones representing the 3'-end of the HGV genome were obtained by amodified anchored RT-PCR method. Briefly, poly A polymerase (GIBCO/BRL,Gaithersburg, Md.) was used to catalyze the addition of a poly(A) tailto PNF 2161 RNA prior to cDNA synthesis. The poly(A) addition wasperformed according to the manufacturer's recommendations. Followingpurification of the poly(A) modified RNA, reverse transcription with"SUPERSCRIPT II" (GIBCO/BRL) was carried out using primer GV-5446IRT(SEQ ID NO:184). The resulting cDNA was amplified by PCR using thefollowing primer set: GV59-5446F (SEQ ID NO:171) and GV-5446IR (SEQ IDNO:172).

After amplification, the products were separated by agarose gelelectrophoresis, transferred to filters and hybridized with adigoxigenin-labelled oligonucleotide probe (E5-7-PRB, SEQ ID NO:173).Products that hybridized with the oligonucleotide were isolated,purified, cloned into "pCR-II" and sequenced. The two clones isolated bythis method were MP3-3 (SEQ ID NO:168) and MP3-7 (SEQ ID NO:169).

EXAMPLE 7 Isolation of 470-20-1 Fusion Protein

A. Expression and Purification of 470-20-1/Glutathione-S-TransferaseFusion Protein

Expression of a glutathione-S-transferase (sj26) fused proteincontaining the 470-20-1 peptide was achieved as follows. A 237 base pairinsert (containing 17 nucleotides of SISPA linkers on both sides)corresponding to the original lambda gt11 470-20-1 clone was isolatedfrom the lambda gt11 470-20-1 clone by polymerase chain reaction usingprimers gt11 F(SEQ ID NO:25) and gt11 R(SEQ ID NO:13) followed by Eco RIdigestion.

The insert was cloned into a modified pGEX vector, pGEX MOV. pGEX MOVencodes sj26 protein fused with six histidines at the carboxy terminalend (sj26his). The 470-20-1 polypeptide coding sequences were introducedinto the vector at a cloning site located downstream of sj26his codingsequence in the vector. Thus, the 470-20-1 polypeptide is expressed assj26his/470-20-1 fusion protein. The sj26 protein and six histidineregion of the fusion protein allow the affinity purification of thefusion protein by dual chromatographic methods employingglutathione-conjugated beads (Smith, D. B., et al.) and immobilizedmetal ion beads (Hochula; Porath).

E. coli strain W3110 (ATCC catalogue number 27352) was transformed withpGEX MOV and pGEX MOV containing 470-20-1 insert. Sj26his protein and470-20-1 fusion protein were induced by the addition of 2 mMisopropyl-β-thiogalactopyranoside (IPTG). The fusion proteins werepurified either by glutathione-affinity chromatography or by immobilizedmetal ion chromatography (IMAC) according to the published methods(Smith, D. B., et al.; Porath) in conjunction with conventionalion-exchange chromatography.

The purified 470-20-1 fusion protein was immunoreactive with PNF 2161.However, purified sj26his protein was not immunoreactive with PNF 2161,indicating the presence of specific immunoreaction between the 470-20-1peptide and PNF 2161.

B. Isolation of 470-20-1/B-Galactosidase Fusion Protein

KM392 lysogens infected either with lambda phage gt11 or withgt11/470-20-1 are incubated in 32° C. until the culture reaches to anO.D. of 0.4. Then the culture is incubated in a 43° C. water bath for 15minutes to induce gt11 peptide synthesis, and further incubated at 37°C. for 1 hour. Bacterial cells are pelleted and lysed in lysis buffer(10 mM Tris, pH 7.4, 2% "TRITON X-100" and 1% aprotinin). Bacteriallysates are clarified by centrifugation (10K, for 10 minutes, SorvallJA20 rotor) and the clarified lysates are incubated with Sepharose 4Bbeads conjugated with anti-β-galactosidase (Promega).

Binding and elution of β-galactosidase fusion proteins are performedaccording to the manufacturer's instruction. Typically binding of theproteins and washing of the column are done with lysis buffer. Boundproteins are eluted with 0.1M carbonate/bicarbonate buffer, pH 10. Thepurified 470-20-1/b-galactosidase protein is immunoreactive with bothPNF2161 and anti-b-galactosidase antibody. However, β-galactosidase,expressed by gt11 lysogen and purified, is not immunoreactive withPNF2161 but immunoreactive with anti-β-galactosidase antibody.

EXAMPLE 8 Purification of the 470-20-1 Fusion Protein and Preparation ofAnti-470-20-1 Antibody

A. Glutathione Affinity Purification

Materials included 50 ml glutathione affinity matrix reduced form(Sigma), XK 26/30 Pharmacia column, 2.5×10 cm Bio-Rad "ECONO-COLUMN"(Richmond, Calif.), Gilson (Middleton, Wis.) HPLC, DTT (Sigma),glutathione reduced form (Sigma), urea, and sodium phosphate dibasic.

The following solutions were used in purification of the fusion protein:

Buffer A: phosphate buffer saline, pH 7.4, and

Buffer B: 50 mM Tris Ph 8.5, 8 mM glutathione, (reduced formglutathione)

Strip buffer: 8M urea, 100 mM Tris pH 8.8, 10 mM glutathione, 1.5 NaCl.

E. coli carrying the plasmid pGEX MOV containing 470-20-1 insert, weregrown in a fermentor (20 liters). The bacteria were collected and lysedin phosphate buffered saline (PBS) containing 2 mM phenylmethyl sulfonylfluoride (PMSF) using a micro-fluidizer. Unless otherwise noted, all ofthe following procedures were carried out at 4° C.

The crude lysate was prepared for loading by placing lysed bacteria into"OAKRIDGE" tubes and spinning at 20K rpms (40k ×g) in a Beckman modelJA-20 rotor. The supernatant was filtered through a 0.4 μm filter andthen through a 0.2 μm filter.

The 2.5×10 cm "ECONO-COLUMN" was packed with the glutathione affinitymatrix that was swelled in PBS for two hours at room temperature. Thecolumn was brought into equilibrium by washing with 4 bed volumes ofPBS.

The column was loaded with the crude lysate at a flow rate of 8 ml perminute. Subsequently, the column was washed with 5 column volumes of PBSat the same flow rate.

The column was eluted by setting the flow rate to 0.75-1 ml/min. andintroducing Buffer B. Buffer B was pumped through the column for 5column volumes and two-minute fractions were collected. An exemplaryelution profile is shown in FIG. 2. The content and purity of theproteins present in the fractions were assessed by standard SDS PAGE(FIG. 3). The 470-20-1/sj26his fusion protein was identified based onits predicted molecular weight and its immunoreactivity to PNF 2161serum. For further manipulations, the protein can be isolated fromfractions containing the fusion protein or from the gel by extraction ofgel regions containing the fusion protein.

B. Purification of Clone 470-20-1 Fusion Protein by Anion Exchange.

Solutions include the following:

Buffer A (10 mM sodium phosphate pH 8.0, 4M urea, 10 mM DTT);

Buffer B (10 mM sodium phosphate pH 8.0, 4M urea, 10 mM DTT, 2.0M NaCl);and

Strip Buffer (8M urea, 100 mM Tris pH 8.8, 10 mM glutathione, 1.5 NaCl).

Crude lysate (or other protein source, such as pooled fractions fromabove) was loaded onto "HIGH-Q-50" (Biorad, Richmond, Calif.) column ata flow rate of 4.0 ml/min. The column was then washed with Buffer A for5 column volumes at a flow rate of 4.0 ml/min.

After these washes, a gradient was started and ran from Buffer A toBuffer B in 15 column volumes. The gradient then stepped to 100% BufferB for one column volume. An exemplary gradient is shown in FIG. 4A.Fractions were collected every 10 minutes. Purity of the470-20-1/sj26his fusion protein was assessed by standard SDS-PAGE (FIGS.4B and 4C) and relevant fractions were pooled (approximately fractions34 through 37, FIG. 4C).

C. Preparation of Anti-470-20-1 Antibody

The purified 470-20-1/sj26his fusion protein is injected subcutaneouslyin Freund's adjuvant in a rabbit. Approximately 1 mg of fusion proteinis injected at days 0 and 21, and rabbit serum is typically collected at6 and 8 weeks.

A second rabbit is similarly immunized with purified sj26his protein.

Minilysates are prepared from bacteria expressing the 470-20-1/sj26hisfusion protein, sj26his protein, and β-galactosidase/470-20-1 fusionprotein. The lysates are fractionated on a gel and transfered to amembrane. Separate Western blots are performed using the sera from thetwo rabbits.

Serum from the animal immunized with 470-20-1 fusion protein isimmunoreactive with all sj26his fusion protein in minilysates of IPTGinduced E. coli W3110 that are transformed either with PGEX MOV or withpGEX MOV containing 470-20-1 insert. This serum is also immunoreactivewith the fusion protein in the minilysate from the 470-20-1 lambda gt11construct.

The second rabbit serum is immunoreactive with both sj26his and470-20-1/sj26his fusion proteins in the minilysates. This serum is notexpected to immunoreactive with 470-20-1/β-galactosidase fusion proteinin the minilysate from the 470-20-1 lambda gt11 construct. None of thesera are expected to be immunoreactive with β-galactosidase.

Anti-470-20-1 antibody present in the sera from the animal immunizedwith the fusion protein is purified by affinity chromatography (usingthe 470-20-1 ligand).

Alternatively, the fusion protein can be cleaved to provide the 470-20-1antigen free of the sj-26 protein sequences. The 470-20-1 antigen aloneis then used to generate antibodies as described above.

EXAMPLE 9 Rabbit Anti-Peptide Sera

Peptides were designed to cover the entire HGV sequence, in particular,to cover each of the functional groups in the non-structural andstructural genes. Peptides were synthesized commercially by conventionaltechniques. Representative peptides are presented in Table 12.

                  TABLE 12                                                        ______________________________________                                                       Size of                                                                       Peptide End Points Relative                                    Desgination    (aa)    to SEQ ID NO:14                                        ______________________________________                                        PEP1/NS2a      30      2674/2763                                              PEP2/E1        16      733/780                                                PEP3/E2        18      1219/1272                                              PEP4/NS2B      18      3061/3114                                              PEP5/NS3       21      3571/3633                                              PEP6/NS3**     18      4909/4959                                              PPE7/NS4A      18      5275/5328                                              PEP8/NS4B      16      6097/6144                                              PEP9/NS5A      16      7033/7080                                              PEP10/NS5B     18      7783/7836                                              ______________________________________                                         **The NS3 peptide has an extraneous Cysteine on the C terminal end that i     not in the HGVPNF 2161 variant polypeptide sequence; the actual sequence      was a Q.                                                                 

The peptides were coupled to KLH. Using rabbits as host, the conjugatedpeptides were injected subcutaneously at multiple sites. Anti-peptiderabbit serum were generated by a commercial facility. A two-weekimmunization protocol was used with bleeds taken at alternate weeks.

Rabbit anti-peptide sera were shown to be peptide specific and to havehigh titer. Rabbit anti-peptide sera also recognize correspondingrecombinant proteins expressed in E. coli and baculovirus. Antibodyendpoint titers range from 1:50,000 dilution to 1:625,000 dilution.Rabbit anti-peptide 7 (NS4a) had low end point titers of only 1:1,000.Accordingly, rabbit anti-serum to the NS4a protein expressed in, forexample, the baculovirus system may be a more useful reagent.

Rabbit anti-peptide sera are useful for immunoprecipitatingcorresponding HGV proteins expressed, for example, in baculovirus andvaccinia. Rabbit anti-peptide sera are also useful as capture antibodyin EIAs to detect HGV antigen. Rabbit anti-peptide sera are furtheruseful in the characterization of the HGV proteins.

EXAMPLE 10 Serology

A. Western Blot analysis of Sera Panels

The 470-20-1 fusion antigen (described above) was used to screen panelsof sera. Many of the panels were of human sera derived both fromindividuals suffering from hepatitis and uninfected controls.

Affinity purified 470-20-1 fusion antigen (Example 8) was loaded onto a12% SDS-PAGE at 2 μg/cm. The gel was run for two hours at 200V. Theantigen was transfered from the gel to a nitrocellulose filter.

The membrane was then blocked for 2 hours using a solution of 1% bovineserum albumin, 3% normal goat serum, 0.25% gelatin, 100 mM NaPO₄, 100 mMNaCl, and 1% nonfat dry milk. The membrane was then dried and cut into1-2 mm strips; each strip contained the 470-20-1 fusion antigen. Thestrip was typically rehydrated with TBS (150 mM NaCl; 20 mM Tris HCl, pH7.5) and incubated in panel sera (1:100) overnight with rocking at roomtemperature.

The strips were washed twice for five minutes each time in TBS plus"TWEEN 20" (0.05%), and then washed twice for five minutes each time inTBS. The strips were then incubated in secondary antibody (Promegaanti-human IgG-Alkaline Phosphatase conjugate, 1:7500), for 1 hour withrocking at room temperature. The strips were then washed twice×5 minutesin TBS+"TWEEN 20", then twice×5 minutes in TBS.

Bound antibody was detected by incubating the strips in a substratesolution containing BCIP (Example 2) and NBT (Example 2) in pH 9.5buffer (100 mM Tris, 100 mM NaCl, 5 mM MgCl₂). Color development wasallowed to proceed for approximately 15 minutes at which point colordevelopment was halted by 3 washes in distilled H₂ O.

Test sera were derived from the following groups of individuals: (i)blood donors, negative for HBV Ab, surface Ag, negative for HCV, HIV,HTLV-1 Abs; (ii) HBV, sera from individuals who are infected withHepatitis B virus; (iii) HCV, sera from individuals infected withHepatitis C virus by virtue of being reactive in a second-generation HCVELISA assay; and (iv) HXV, individuals serologically negative for HAV,HBV, HCV, or HEV.

The results of these screens are presented in Table 13.

                  TABLE 13                                                        ______________________________________                                        470-20-1 Sera Panelling Result Summary                                                No. Human*                                                            Sample  Sera Tested                                                                             +         IND*    -                                         ______________________________________                                        blood donor                                                                           30         1 (3.3%)  2 (6.7%)                                                                             27 (90.0%)                                HBV     40         7 (17.5%)                                                                               4 (10.0%)                                                                            29 (72.5%)                                HCV     38        11 (28.95%)                                                                             11 (28.95%)                                                                           16 (42.1%)                                HXV     122       20 (16.4%)                                                                              12 (9.8%)                                                                             90 (73.8%)                                ______________________________________                                         *Indeterminate, weak reactivity                                          

These results suggest the presence of the 470-20-1 antigen in a numberof different sera samples. The antigen is not immunoreactive with normalhuman sera.

B. General Elisa Protocol for Detection of Antibodies

Polystyrene 96 well plates ("IMMULON II" (PGC)) are coated with 5 μg/ml(100 μL per well) antigen in 0.1M sodium bicarbonate buffer, pH 9.5.Plates are sealed with "PARAFILM" and stored at 4° C. overnight.

Plates are aspirated and blocked with 300 uL 10% normal goat serum andincubated at 37° C. for 1 hr.

Plates are washed 5 times with PBS 0.5% "TWEEN-20".

Antisera is diluted in 1×PBS, pH 7.2. The desired dilution(s) ofantisera (0.1 mL) are added to each well and the plate incubated 1 hourat 37° C. The plates are then washed 5 times with PBS 0.5% "TWEEN-20".

Horseradish peroxidase (HRP) conjugated goat anti-human antiserum(Cappel) is diluted 1/5,000 in PBS. 0.1 mL of this solution is added toeach well. The plate is incubated 30 min at 37° C., then washed 5 timeswith PBS.

Sigma ABTS (substrate) is prepared just prior to addition to the plate.

The reagent consists of 50 ml 0.05M citric acid, pH 4.2, 0.078 ml 30%hydrogen peroxide solution and 15 mg ABTS. 0.1 ml of the substrate isadded to each well, then incubated for 30 min at room temperature. Thereaction is stopped with the addition of 0.050 mL 5% SDS (w/v). Therelative absorbance is determined at 410 nm.

EXAMPLE 11 Expression of Selected HGV Antigens

The entire coding sequence of HGV was subcloned into greater than 50distinct overlapping cDNA fragments. The length of most cDNA fragmentsranged from about 200 bp to about 500 bp. The cDNA fragments were clonedseparately into the expression vector, pGEX-HisB. This vector is similarto pGEX-MOV, described above.

pGEX-hisB is a modification of pGEX-2T (Genbank accession number A01438;a commercially available expression vector). The vector pGEX-2T has beenmodified by insertion of a NcoI site directly downstream from thethrombin cleavage site. This site is followed by a BamHI site, which isfollowed by a poly-histidine (six histidines) encoding sequence,followed by the EcoRI site found in pGEX-2T. Coding sequences ofinterest are typically inserted between the NcoI site and the BamHIsite. In FIG. 6 (SEQ ID NO:115), the inserted sequence encodes the GE3-2antigen. The rest of the vector sequence is identical to pGEX-2T.Expression of fusion protein is carried out essentially as describedabove with other pGEX-derived expression vectors.

Cloning of all 50 fragments was carried out essentially as describedbelow, where specific primers were selected for each of the 50 codingregions. Each HGV insert DNA is PCR amplified from RNA extracted fromPNF 2161 or other HGV(+) sera using a specific set of primers asdescribed in Example 4C. Typically, the 5' primer contained a NcoIrestriction site and the 3' primer contained a BamHI restriction site.The NcoI primers in the amplified fragments allowed in-frame fusion ofamplified coding sequences to the GST-Sj26 coding sequence in theexpression vectors pGEX-Hisb or pGEX MOV.

Amplified HGV insert DNA is digested with restriction enzymes NcoI andBam HI. Digested insert DNA is gel purified and ligated with NcoI andBamHI digested pGEX hisB or pGEX MOV. E. coli strain W3110 (ATCC #27325,American Type Culture Collection, Rockville, Md.) was transformed withthe ligation product. Ampicillin resistant colonies were selected.Presence of the insert was confirmed by the PCR amplification of theinsert from the ampicillin resistant colony using primers homologous toPGEX vector sequences flanking the inserted molecules (primers GLI F(SEQ ID NO:235) and GLI R (SEQ ID NO:236).

The size of the PCR amplification product is the insert size plusapproximately 160 bp derived from vector. Transformants with appropriateinserts were selected and subjected to protein induction by IPTG asdescribed in Example 7. Expressed recombinant proteins were analyzed forspecific immunoreactivity against putative HGV-infected human sera byWestern blot.

Eight fragments designated GE3, GE9, GE15, GE17, GE4, EXP3, GE1-N andGE-57 encoded antigens that gave a clear immunogenic response whenreacted with putative HGV-infected human sera.

A. CLONING OF GE3, GE9, GE15, GE17, GE4, EXP3, GE1-N AND GE57.

The coding sequence inserts for clones GE3, GE9, GE15, GE17, GE4, EXP3,GE1-N and GE57 were generated by polymerase chain reaction fromSISPA-amplified double-stranded cDNA or RNA obtained from PNF 2161 orT55806 using PCR primers specific for each fragment. Following Table 14lists the coordinates of each clone relative to SEQ ID NO:14 and theprimer sets used for generation of each clone insert.

                  TABLE 14                                                        ______________________________________                                             Serum    Coordinate on                                                                            F Primer  R Primer                                   Clone                                                                              Source   SEQ ID NO:14                                                                             (SEQ ID NO:)                                                                            (SEQ ID NO:)                               ______________________________________                                        GE3  PNF 2161 6615-6977  GE-3F     GE-3R                                                               (SEQ ID NO:46)                                                                          (SEQ ID NO:47)                             GE9  PNF 2161 8154-8441  GE-9F     GE-9R                                                               (SEQ ID NO:48)                                                                          (SEQ ID NO:49)                             GE15 PNF 2161 3615-3935  GE-15F    GE-15R                                                              (SEQ ID NO:111)                                                                         (SEQ ID NO:112)                            GE17 PNF 2161 3168-3305  GE-17F    GE-17R                                                              (SEQ ID NO:113)                                                                         (SEQ ID NO:114)                            GE4  PNF 2161 6825-7226  GE4F      GE4R                                                                (SEQ ID NO:149)                                                                         (SEQ ID NO:150)                            EXP3 PNF 2161 6648-7658  470EXP3F  470EXP3R                                                            (SEQ ID NO:151)                                                                         (SEQ ID NO:152)                            GE1- PNF 2161 5850-6239  GE1-NF    GE1-NR                                     N                        (SEQ ID NO:237)                                                                         (SEQ ID NO:238)                            GE57 T55806   271*-456*  GE57F     GE57R                                                               (SEQ ID NO:239)                                                                         (SEQ ID NO:240)                            ______________________________________                                         *These sequences are given relative to SEQ ID NO:178.                    

The amino acid sequence of GE57 is presented as SEQ ID NO:241.

In the GE3-5' primer (GE-3F, SEQ ID NO:46) a silent point mutation wasintroduced to modify a natural NcoI restriction site. Using theabove-described primers, PCR amplification products were generated. Theamplification products were gel purified, digested with NcoI and BamHI,and gel purified again. The purified NcoI/BamHI GE3, GE9, GE15, GE17,GE4, GE1-N and GE57 fragments were independently ligated intodephosphorylated, NcoI/BamHI cut pGEX-HisB vectors. The purifiedNcoI/BamHI EXP3 fragment was ligated into dephosphorylated, NcoI/BamHIcut PGEX-MOV vector.

Each ligation mixture was transformed into E. coli W3110 strain andampicillin resistant colonies were selected. The ampicillin resistantcolonies were resuspended in a Tris/EDTA buffer and analyzed by PCR,using primers GLI F (SEQ ID NO:235) and GLI R (SEQ ID NO:236) to confirmthe presence of insert sequences. Eight candidate clones were designatedGE3-2, GE9-2, GE15-1, GE17-2, GE4-8, EXP3-7, GE1-N and GE57,respectively.

B. Expression of the GE3-2, GE9-2, GE15-1, GE17-2, GE4-8, EXP3-7, GE1-Nand GE57 Fusion Proteins.

Colonies of ampicillin resistant bacteria carrying GE3-2, GE9-2, GE15-1,and GE17-2, GE4-8, EXP3-7, GE1-N and GE57 containing-vectors wereindividually inoculated into LB medium containing ampicillin. Thecultures were grown to OD of 0.8 to 0.9 at which time IPTG(isopropylthio-beta-galactoside; Gibco-BRL) was added to a finalconcentration of 0.3 to 1 mM, for the induction of protein expression.Incubation in the presence of IPTG was continued for 3 to 4 hours.

Bacterial cells were harvested by centrifugation and resuspended in SDSsample buffer (0.0625M Tris, pH 6.8, 10% glycerol, 5% mercaptoethanol,2.3% SDS). The resuspended pellet was boiled for 5 min. and then clearedof insoluble cellular debris by centrifugation. The supernatantsobtained from IPTG-induced cultures of GE3-2, GE9-2, GE15-1, GE17-2,GE4-8, EXP3-7, GE1-N and GE57 were analyzed by SDS-polyacrylamide gelelectrophoresis (PAGE) together with uninduced lysates. The proteinsfrom these gels were then transferred to nitrocellulose filters (i.e.,by Western blotting).

The filters were first incubated with rabbit polyclonal antibody ormouse monoclonal antibody (RM001 from Sierra Biosource, Calif.) directedto GST protein to detect the expression of appropriate size GST-fusionprotein expression. Expected protein sizes of above clones are 40, 38,39, 32, 42, 64, 42 and 33 KDa, respectively. Immunoreactivity of RM001with bands at the appropriate molecular weight for the fusion proteinsdemonstrated the successful expression of the fusion proteins of aboveclones by the bacterial cells. Expression of the clone proteins werealso monitored by the appearance of over-expressed proteins ofappropriate sizes upon IPTG induction on the Coomassie brilliant bluestained gel.

C. Western Blot Analysis of HGV Proteins.

Once the expression of the HGV clone protein was confirmed by Westernblot analysis with anti-GST antibody a second set of filters, preparedas above, were then exposed to several HGV(+) and HGV(-) human sera.Human sera used for Western blot analyses of whole cell lysates werepre-absorbed with the lambda-gt11-nitrocellulose filters.Lambda-gt11-nitrocellulose filters were prepared as follows. Briefly, anovernight culture of KM392 culture was prepared in LB. The culture wasdiluted 10 fold in fresh LB containing 0.2% maltose and incubated for 1hour at 37° C. with shaking.

After 1 hour the culture was mixed with an equal volume of MgCa solution(0.01M MgCl₂ and 0.01M CaCl₂). To this mixture lambda gt11 was added toa titer of 2×10⁴ PFU/ml and incubated for 30 min without shaking. After30 minutes (per each ml of this phage/E.coli mixture) 15 ml of molten(55° C.) LB top agar (LB with 0.8% agar) was added: 8 ml of this mixturewas spread onto each 15 cm LB agar plate. After the top agar solidifiedthe plate was incubated at 37° C. for 3-5 hr.

After plaques developed, a nitrocellulose filter was placed on the plateand the plate further incubated at 37° C. overnight. The nitrocellulosefilter was removed and washed thoroughly with TBS (50 mM Tris-HCl, pH7.5, 150 mM NaCl) plus 0.05% "TWEEN 20." The washed filter was thenblocked with 1% gelatin in TBS overnight. The filter was washed threetimes (5 minutes each wash) with TBS.

For the pre-absorption of human sera each serum was diluted 100 fold inblocking solution (described in Example 10). Ten mls. of diluted serumwas then incubated overnight with two lambda gt11 filters prepared asabove. Lambda gt11 filters were removed and the pre-absorbed serum usedfor Western blot analysis.

Western blot analyses demonstrated that clones GE3-2, GE9-2, GE15-1,GE17-2, GE4-8, EXP3-7, GE1-N and GE57 showed specific immunoreactivitytoward HGV(+) sera. The GE-4-8 protein was immunoreactive with J21689serum. J21689 is HGV (+) serum as determined by HGV PCR (Example 4) andHCV (+) as determined by HCV PCR and serological analyses. The EXP3-7protein was immunoreactive with JC and T55806. JC is the HGV-positiveserum identified in Example 4F that was rejected by the blood bank forbeing high ALT. A second JC sample, taken one year after the initialserum sample, was also positive for HGV by PCR analysis. T55806 is alsothe HGV-positive serum identified in Example 4F that was rejected by theblood bank for being High ALT. This serum is co-positive with HCV.

Further, GE15-1 and GE-17 showed weak but specific immunoreactivitytoward PNF 2161 and T55806. GE1-N was immunoreactive with PNF2161, JC,T55806, T56633, T27034 and R0001. T56633, T27034 and R0001 are HGV (+)sera identified in Example 4F. GE57 was immunoreactive with E57963 andR0001. E57963 is HGV and HCV co-positive serum. GE3-2 and GE9-2 werealso immunoreactive with HGV sera specifically. However, none of theeight antigens were immunoreactive with HGV negative sera T43608 andR05072.

The GE3-2 and GE9-2 fusion proteins were purified from bacterial celllysates essentially as in Example 7 using dual chromatographic methodsemploying glutathione-conjugated beads (Smith, D. B., et al.) andimmobilized metal ion beads (Hochuli; Porath). The purified proteinswere subjected to Western blot analysis as follows.

Various amounts of the purified HGV proteins (e.g., GE3-2 and GE9-2proteins) were loaded on 12% acrylamide gels. Following PAGE, proteinswere transferred from the gels to nitrocellulose membranes, usingstandard procedures. Individual membranes were incubated with one of anumber of human or mouse sera. Excess sera were removed by washing themembranes.

These membranes were incubated with alkaline phosphatase-conjugated goatanti-human antibody (Promega) or alkaline phosphatase-conjugated goatanti-mouse antibodies (Sigma), depending on the serum being used forscreening. The membranes were washed again, to remove excess goatanti-human IgG antibody, and exposed to NBT/BCIP. Photographs ofexemplary stained membranes having the GE3 fusion protein are shown inFIGS. 7A to 7D.

The Figures show the results of Western blot analysis of the purifiedGE3-2 protein using the following sera: N-(ABCDE) human (JC) serum (FIG.7A), N-(ABDE) human (PNF 2161) serum (FIG. 7B), a super normal (SN2)serum (FIG. 7C), and mouse monoclonal antibody (RM001) directed againstGST-Sj26 protein (FIG. 7D).

In each of the figures, lane 1 contains pre-stained molecular weightstandards(Bio-Rad), and lanes 2-5 contain, respectively, the followingamounts of the GE3-2 fusion protein: 4 μg, 2 μg, 1 μg, and 0.5 μg.Numbers represent loading amounts in micrograms per 0.6 centimeter ofgel (well size). Dilutions of the human JC, PNF 2161 and Super Normal 2sera were 1:100. The anti-sj26 dilution was 1:1000. The band seen atabout 97K in the JC blot is reactivity against a minor contaminant inthe GE3.2 fusion protein preparation. Protein marker sizes are 142.9,97.2, 50, 35.1, 29.7 and 21.9 KD.

As shown in FIGS. 7A to 7D, GE3-2 showed specific immunoreactivity withJC serum. GE3-2 reacted weakly with PNF 2161 serum and would be scoredas an indeterminant or negative.

In parallel experiments, GE9-2 showed weak but specific immunoreactivitytoward PNF 2161 serum.

EXAMPLE 12 Construction of Exemplary Epitope Libraries

A. The Y5 Library.

Polymerase Chain Reactions were employed to amplify 3 overlapping DNAfragments from PNF 2161 SISPA-amplified cDNA. The PNF 2161SISPA-amplified cDNA was prepared using the JML-A/B linkers (SEQ IDNO:54 and SEQ ID NO:55). One microliter of this material wasre-amplified for 30 cycles (1 minute at 94° C., 1.5 minutes at 55° C.and 2 minutes at 72° C.) using 1 μM of the JML-A primers. The totalreaction volume was 100 μl. The products from 3 of these amplificationswere combined and separated from excess PCR primers by a single passthrough a "WIZARD PCR COLUMN" (Promega) following the manufacturer'sinstructions. The "WIZARD PCR COLUMN" is a silica based resin that bindsDNA in high ionic strength buffers and will release DNA in low ionicstrength buffers. The amplified DNA was eluted from the column with 100μl distilled H20.

The eluted DNA was fractionated on a 1.5% Agarose TBE gel (Maniatis, etal.) and visualized with UV light following ethidium bromide staining. Astrong smear of DNA fragments between 150 and 1000 bp was observed. Onemicroliter of the re-amplified cDNA was used as for template in PCRreactions with each primer pair presented in Table 15.

                  TABLE 15                                                        ______________________________________                                        Primers    SEQ ID NO: Size of Amplified Fragment                              ______________________________________                                        470ep-F1   SEQ ID NO:56                                                                             810                                                     470ep-R1   SEQ ID NO:57                                                       470ep-F2   SEQ ID NO:58                                                                             750                                                     470ep-R3   SEQ ID NO:59                                                       470ep-F4   SEQ ID NO:60                                                                             669                                                     470ep-R4   SEQ ID NO:61                                                       ______________________________________                                    

The primers were designed to result in the amplification of HGV specificDNA fragments of the sizes indicated in Table 15. In the amplificationreactions, the primer pairs were used at a concentration of 1 μM.Amplifications were for 30 cycles of 1 minute at 94, 1.5 minutes at 54°C. and 3 minutes at 72° C. in a total reaction volume of 100 μl. Each ofthe three different primer pair PCR reactions resulted in the specificamplification of products having the expected sizes. For each primerpair reaction, amplification products from 3 independent PCR reactionswere combined and purified using a "WIZARD PCR COLUMN" as describedabove. The purified products were eluted in 50 μl dH20.

Samples from each purified product (14 μl, containing approximately 1-2μg of each primer-pair amplified DNA fragment) were combined. Thecombined sample of all three different amplified fragments was added to5 μl of 10× DNAse Digestion buffer (500 mM Tris PH 7.5, 100 mM MnCl₂)and 2 μl of dH20. From this digestion mixture, a 10 μl sample wasremoved and placed in a tube containing 5 μl of Stop solution (100 mMEDTA, pH 8.0). This sample was the 0 "minutes of digestion" time point.The rest of the digestion reaction was placed at 25° C. To the digestionmixture 1 μl of 1/25 diluted RNase-free DNAse I (Stratagene) was added.At various time points 10 μl aliquots were withdrawn and mixed with 5 μlof Stop solution. The DNAse I digested DNA products were analyzed on a1.5% Agarose TBE gel.

The results of several digestion experiments showed that 40 minutes ofdigestion provided a good distribution of DNA fragments in the sizerange of 100-300 bp. A DNAse I digestion was then repeated with theentire digestion being left for 40 minutes at room temperature. Thedigestion was stopped by the addition of 18 μl of Stop Buffer and thedigested DNA products were purified using a "WIZARD PCR COLUMN." The"WIZARD-PCR COLUMN" was eluted with 50 μl of dH20 and the eluted DNAadded to the following reaction mixture: 7 μl of Restriction EnzymeBuffer C (Promega, 10 mM MgCl₂, 1 mM DTT, 50 mM NaCl, 10 mM Tris, pH7.9, 1× concentration); 11 μl of 1.25 mM dNTPs; and 2 μl T4 DNAPolymerase (Boehringer-Mannhiem). This reaction mixture was held at 37°C. for 30 minutes, at which point 70 μl of pH 8.0 phenol/CHCl₃ was addedand mixed. The phenol/CHCl₃ was removed and extracted once to yield atotal aqueous volume of 150 μl containing the DNA sample. The DNA wasethanol precipitated using 2 volumes of absolute ethanol and 0.5 volumeof 7.5M NH₄ -acetate. The DNA was pelleted by centrifugation for 15minutes at 14,000 rpm in an "EPPENDORF MICROFUGE", dried for 5 minutesat 42° C. and resuspended in 25 μl of dH20.

The DNA was ligated to 5' phosphorylated SISPA linkers KL1 (SEQ IDNO:62) and KL2 (SEQ ID NO:63). Several different concentrations of SISPAlinkers and DNA was tested. The highest level of ligation (assessed asdescribed below) occurred under the following ligation reactionconditions: 6 μl of DNA, 2 μl of 5.0×10-12M KL1/KL2 linkers, 1 μl of 10×ligase buffer (New England Biolabs), and 1 μl of 400 Units/μl T4 DNALigase (New England Biolabs) in a total reaction volume of 10 μl.Ligations were carried out overnight at 16° C.

Two reactions were run in parallel as follows. A 2 μl sample of theligated material was amplified using the KL1 SISPA primer in a totalreaction volume of 100 μl (25 cycles of 1 minute at 94° C., 1.5 minutesat 55° C. and 2 minutes at 72° C.). The degree of ligation was assessedby separating 1/5 of the PCR reaction amplified products byelectrophoresis using a 1.5% agarose TBE gel. The gel was stained withethidium bromide and the bands visualized with UV light.

The amplification products from the duplicate reactions were purifiedusing "WIZARD PCR COLUMNS" and the purified DNA eluted in 50 μl of dH20.A twenty-five microliter aliquot of the PCR KL1/KL2 amplified DNA wasdigested with 36 Units of EcoRI (Promega) in a total volume of 30 μl.The reaction was carried out overnight at 37° C. The Digested DNA waspurified using a "SEPHADEX G25" spin column.

The EcoRI digested DNA was ligated in overnight reactions to λgt11 armsthat were pre-digested with EcoRI and treated with calf intestinalalkaline phosphatase (Stratagene, La Jolla, Calif.). The ligationmixture was packaged using a "GIGAPACK GOLD PACKAGING EXTRACT"(Stratagene) following manufacturer's instructions. Titration of theamount of recombinant phage obtained was performed by plating a 1/10dilution of the packaged phage on a lawn of KM-392, where the platecontained 20 μl of a 100 mg/ml solution of x-gal(5-Bromo-4-chloro-3-indolyl-β-D-galactoside; Sigma) and 20 μl of a 0.1Msolution of IPTG (Isopropyl-1-thio-β-D-galactoside; Sigma). A titer wasobtained of 1.2×10⁶ phage/ml containing over 75% recombinant phage.

The percentage of recombinant plaques was confirmed by PCR analysis of 8randomly picked plaques using primers 11F (SEQ ID NO:25) and 11R (SEQ IDNO:13). This packaged library containing the DNA fragments derived fromthe digestion of the amplified DNAs F1/R1, F2/R3, and F4/R4 amplifiedDNAs and was designated library Y5.

B. The ENV Library.

An expression library, designated the ENV library, was generated asfollows. One microliter of PNF 2161 SISPA amplified DNA was used as thetemplate in polymerase chain amplification reactions utilizing thefollowing primer pairs: GEP-F15 (SEQ ID NO:128) and GEP-R15 (SEQ IDNO:129), which generate a 525 nucleotide HGV fragment; and GEP-F17 (SEQID NO:130) and GEP-R16 (SEQ ID NO:131), which generate a 765 nucleotideHGV fragment.

PCR amplification was for 35 cycles of 94° C. for 1 min, 52° C. for 1.5minutes, and 72° C. for 3 minutes. The amplified products were purifiedand digested with DNAse I. Ligation of KL1 and KL2 linkers to cDNA,amplification of DNA fragments and construction of libraries in lambdagt11 were performed essentially as described in Example 12A. Therecombinant frequency of the library was greater than 70%. Analysis ofthe inserts by polymerase chain reaction using primers derived from theflanking regions of lambda gt11 confirmed the recombinant frequency andindicated that the insert size range was 150-500 nucleotides.

C. The NS3 Library.

An expression library designated NS3 was constructed as follows. A firstfragment was amplified by polymerase chain reaction using the primers470ep-F9 (SEQ ID NO:132) and 470ep-R9 (SEQ ID NO:133) and, as template,PNF 2161 SISPA amplified nucleic acids. The predicted product of thisamplification reaction was 777 base pairs. The amplified fragment wasgel purified by separation on a TAE gel. The fragment was furtherpurified using "GENECLEAN" (Bio 101, La Jolla, Calif.).

Fragment F9/R9 was also amplified using the extension clone GE3L-11 (SEQID NO:41) as source material. Approximately 25 ng of GE3L-11 was used astemplate with the F9 and R9 primers in amplification reactions.

Both of the F9/R9 amplifications were for 30 cycles of 94° C., for 1minute, 52° C. for two minutes, and 72° C. for 3 minutes, using "TAQSTART" (Clonetech, Palo Alto, Calif.). The amplification products fromboth reactions were combined. The products were digested with DNAse I(10 μl GE3L product and 25 ul of PNF SISPA product). The GE3L-basedamplification product represented the majority of the amplificationproduct starting material. Ligation of KL1 and KL2 linkers to cDNA,amplification of DNA fragments and construction of libraries in lambdagt11 were performed essentially as described in Example 12A.

The titer obtained was 2.5×10⁶ phage/ml and the percent recombinantphage was determined to be greater than 99%. Polymerase chain reactionanalysis of the insert sizes confirmed the recombinant frequency andindicated an insert size range of 150 to 550 nucleotides.

In addition, a second fragment was also amplified using theGEP-F10/GEP-R10 primers (SEQ ID NO:135 and SEQ ID NO:136, respectively).One microliter of PNF 2161 SISPA amplified nucleic acids was used astemplate. The predicted fragment size of 570 nucleotides was obtained.The resulting amplification products were manipulated as just describedfor the F9/R9 amplifications. The titer obtained for this fragment wheninserted in lambda gt11 was 1.47×10⁶ phage/ml, with a recombinantfrequency of 90%.

D. The NS2 Library.

The NS2 epitope library was constructed using the methodologiesdescribed in Example 12A. Four DNA fragments containing all or part ofthe HGV proteins NS2, NS3, and NS5b were amplified from 1 ul of PNF 2161SISPA DNA (prepared essentially as described in Example 12A). Thelibrary was generated using the primers given in Table 16 and SISPAamplified PNF 2162 DNA as template.

                  TABLE 16                                                        ______________________________________                                        Fragments        nt                                                           ______________________________________                                        9E-REV (SEQ ID NO:264)                                                                         592      aa 358 (of 389) of                                  E394-R (SEQ ID NO:265)    E2 to aa 166 of NS-2                                GEP-F12 (SEQ ID NO:266)                                                                        663      aa 144 (of 313) of                                  GEP-R12 (SEQ ID NO:267)   NS-2 to aa 51 of                                                              NS-3                                                GEP-F14 (SEQ ID NO:268)                                                                        715      aa 357-594 of NS-3                                  GEP-R13 (SEQ ID NO:269)                                                       470epF8 (SEQ ID NO:270)                                                                        648      aa 716-847 of NS-5                                  GEP-R14 (SEQ ID NO:271)   (716 to end)                                        ______________________________________                                    

All amplifications were for 35 cycles of 94° C./1 minute, 48° C./2minutes, and 73° C./3 minutes. All amplifications yielded at least afragment of the expected size. The amplified products were mixed and inan approximately 1:1:1:1 ratio and partially digested with DNase I. Asabove, the digestion products were ligated to KL1 SISPA linkers,amplified and EcoRI digested. The digested fragments were ligated intolambda gt11. The ligation reactions were packaged.

The packaged ligation products were plated. The resulting library wasdetermined to contain ˜70% recombinant phage with an observed insertsize of 150 to 500 nucleotides.

E. The VNS5A Library.

Primers 470EXT4-2189R (SEQ ID NO:119) and 470EXT4-29F (SEQ ID NO:120)were used to isolate a 2.1 kb DNA fragment that contains the entirecoding sequences for the HGV proteins NS4b and NS5a, as well as the 3'end of NS4a and the 5' end of NS5b. PCR amplifications using theseprimers were performed as described in Example 4G. Successfulamplification was observed with multiple HGV-infected sera including thefollowing: T56633 was from a blood donor whose donation was rejected dueto an ALT value above the cutoff; samples E21-A and E20 were derivedfrom Egyptian individuals suffering from hepatitis; and sample AH0591 isderived from an Australian individual who developed fulminant hepatitis.

The amplified products of E21-A and E20 were cloned into the T overhangsite of the vector T/A (obtained from InVitrogen, San Diego, Calif.)essentially as described in Example 6. The 2.1 kb HGV inserts from these2 plasmids were then isolated by the digestion of approximately 20 ug ofplasmid DNA with approximately 150 units of the restriction enzymeEcoRI. After incubation overnight at 37° C., the products of thedigestion were separated by TAE agarose gel electrophoresis. Theproducts were excised from the section of the agarose gel containing thefragment of interest. The agarose was melted and extraction of theliberated DNA was carried out using the "GENECLEAN II" kit according tothe manufacturers instructions (Bio 101, La Jolla, Calif.).

The purified 2.1 kb fragments derived from the E21-A and E20 samples, aswell as the DNA fragments obtained from PCR amplification of samplesT56633 and AH0591, were digested separately with DNAse I as described inExample 12A. For all 4 samples digestion conditions were determined thatresulted in the isolation of fragments of between 100 to 1000 nts insize. After purification and trimming (Example 12A) the fragmentsderived from each of the 4 HGV infected samples were ligated separatelyto different sets of SISPA linkers. After ligation the DNAs were SISPAamplified.

The amplified DNAs were separately digested overnight at 37° C. withapproximately 100 units of EcoRI. The digested DNAs were then purifiedby spin column chromatography using G25 resin (5'3' Inc, Boulder,Colo.). Digested DNA from the samples T56633, AH0591, and E21-A werecombined at a ratio of 1:1:1 and the mixture of DNAs was ligated intothe EcoRI site of λgt11 as described in Example 12A. After packagingusing the "GIGAPACK III XL" extract (Stratagene, LaJolla, Calif.), theresulting library was plated in the presence of IPTG and XGAL anddetermined to have a titer of approximately 1.0×10⁶ phage/ml and arecombinant frequency of approximately 70%.

EXAMPLE 13 Immunoscreening of the Epitope Libraries

A. Isolation of Immunoreactive Y5 Clones.

Two HGV positive sera, PNF2161 and JC, were used for immunoscreening ofthe Y5 library, essentially as described in Example 2. The Y5 phagelibrary was plated onto 20 plates at approximately 15,000 phage perplate. The plates were incubated for approximately 5 hours and wereoverlaid with nitrocellulose filters (Schleicher and Schuell) overnight.The filters were blocked by incubation in AIB (1% gelatin plus 0.02% Naazide) for approximately 6 hours. The blocked filters were washed oncewith TBS.

Ten Y5 library filters were incubated overnight, with agitation, withPNF2161 serum and ten filters with JC serum. Both sera were diluted 1:10in AIB. In order to reduce non-specific antibody binding, the dilutedsera had been pre-treated by incubation overnight with nitrocellulosefilters to which wild type λgt11 were adsorbed.

The filters were removed from the sera, washed 3 times with TBS andincubated with goat anti-human alkaline phosphatase-conjugated secondaryantibody (Promega; diluted 1/7500 in AIB) for one hour. The filters werewashed 4 times with TBS. Bound secondary antibody was detected byincubation of the filters in AP buffer (100 mM NaCl, 5 mM MgCl₂, 100 mMTris pH 9.5) containing NBT and BCIP.

Plaques that tested positive in the initial screen were picked andeluted in 500 μl of PDB (100 mM NaCl, 8.1 mM MgSO₄, 50 mM Tris pH 7.5,0.02% Gelatin). The immunoreactive phage were purified by replating theeluted phage at a total density of 100-500 plaques per 100 mm plate. Theplates were re-immunoscreened with the appropriate HGV-positive sera,essentially as described above. After color development severalisolated, positive plaques were picked and put into 500 μl of PDB. After1 hour of incubation, 2 μl of the re-purified phage PDB solution wasused as template in a PCR reaction containing the 11F (SEQ ID NO:25) and11R (SEQ ID NO:13) PCR primers. These primers are homologous tosequences located 70 nucleotides (nt) 5' and 90 nt 3' of the EcoRI siteof λgt11. The PCR reactions were amplified through 30 cycles of 94° C.for 1 minute, 55° C. for 1.5 minutes and 72° C. for 2 minutes.

The PCR amplification reactions were size-fractionated on agarose gels.PCR amplification of purified plaques resulted in a single band for eachsingle-plaque amplification reaction, where the amplified fragmentcontained the DNA insert plus approximately 140 bp of 5' and 3' phageflanking sequences. The amplified products, from PCR reactions resultingin single bands, were purified using a "S-300 HR" spin column(Pharmacia), following manufacturers instructions. The DNA wasquantitated and DNA sequenced employing an Applied Biosystems automatedsequencer 373A and appropriate protocols.

The above-described screening of the Y5 library with JC sera resulted inthe purification and DNA sequencing of the positive-strand clonespresented in Table 17. Positive-strand clones correspond to the 5' to 3'translation of the HGV sequence presented in SEQ ID NO:14--thepolyprotein reading frame.

                  TABLE 17                                                        ______________________________________                                                       Insert   Insert Size                                                                          Nucleic                                                                              Encoded                                       Screening                                                                              Size (base                                                                             (amino Acid SEQ                                                                             Protein SEQ                             Clone Sera     pairs)   acids) ID NO. ID NO.                                  ______________________________________                                        Y5-10 JC       210      62     64     65                                      Y5-12 JC       333      94     66     67                                      Y5-26 JC       303      93     68     69                                      Y5-5  JC       153      36     70     71                                      Y5-3  JC       162      44     72     73                                      Y5-27 JC       288      86     74     75                                      Y5-25 JC       165      36     76     77                                      Y5-20 JC       165      .sup. 19.sup.1                                                                       78     79                                      Y5-16 JC       234      56     80     81                                      ______________________________________                                         .sup.1 the clone contained a double insert, nt 69 to 126 of the clone         insert correspond to HGV sequences.                                      

These clones delineated 2 immunogenic regions within the putative NS5protein of HGV. These two region, relative to the sequence presented asSEQ ID NO:14 are positions 6636 to 6821 and 7278 to 7385.

Further, screening of the Y5 library with PNF 2161 sera resulted in thepurification and DNA sequencing of the following negative-strand clonespresented in Table 18. Negative-strand clones correspond to the 5' to 3'translation of the sequence complementary to the HGV sequence presentedin SEQ ID NO:14.

                  TABLE 18                                                        ______________________________________                                                       Insert   Insert Size                                                                          Nucleic                                                                              Encoded                                       Screening                                                                              Size (base                                                                             (amino Acid SEQ                                                                             Protein                                 Clone Sera     pairs)   acids) ID NO. SEQ ID NO.                              ______________________________________                                        Y5-50 PNF 2161 349      104    82     83                                      Y5-52 PNF 2161 119      .sup. 20.sup.1                                                                       84     85                                      Y5-53 PNF 2161 250      .sup. 33.sup.2                                                                       86     87                                      Y5-55 PNF 2161 143      .sup. 20.sup.3                                                                       88     89                                      Y5-56 PNF 2161 366      110    90     91                                      Y5-57 PNF 2161 231      65     92     93                                      Y5-60 PNF 2161 151      38     94     95                                      Y5-63 PNF 2161 .sup. 125.sup.4                                                                        25     96     97                                      ______________________________________                                         .sup.1 the clone contained a double insert, nt 46 to 105 of the clone         insert correspond to HGV sequences.                                           .sup.2 the clone contained a double insert, nt 19 to 118 of the clone         insert correspond to HGV sequences.                                           .sup.3 the clone contained a double insert, nt 70 to 126 of the clone         insert correspond to HGV sequences.                                           .sup.4 the insert contains an extra, nonHGV sequence between nucleotides      19 and 35.                                                               

All of these sequences contain portions of the original HGV clone470-20-1 isolated using the PNF 2161 serum.

Additional epitope clones from the Y5 library were isolated as follows.The Y5 library was screened with the HGV infected sera J21689 and T56633using the methods described in Example 13. Greater than 400 positiveplaques were obtained, indicating the presence of a strongly immunogenicsequence recognized by both of these HGV infected sera. Ten of thesepositive plaques were purified and DNA sequenced. The results obtainedfrom the DNA sequencing are delineated in Table 19.

                  TABLE 19                                                        ______________________________________                                        CLONE     HGV VAR   SERA      START* STOP                                     ______________________________________                                        Y5-114-1A PNF       J21689    6636   6827                                     Y5-114-2B PNF       J21689    6678   6935                                     Y5-121-19A                                                                              PNF       T56633    6678   7063                                     Y5-121-11A                                                                              PNF       T56633    6636   6917                                     Y5-121-12A                                                                              PNF       T56633    6636   6959                                     Y5-121-15A                                                                              PNF       T56633    6636   6917                                     Y5-121-16A                                                                              PNF       T56633    6636   6989                                     Y5-121-17A                                                                              PNF       T56633    6636   7082                                     Y5-121-20A                                                                              PNF       T56633    6636   6929                                     Y5-121-18A                                                                              PNF       T56633    6636   6896                                     ______________________________________                                         *start/stop locations are given relative to SEQ ID NO:14                 

Comparison of these sequences with those obtained previously fromscreening this library indicated that these clones all contained thesame epitope(s) that are contained in the previously isolated epitopeclone Y5-10. Two of the clones, Y5-114-2B and Y5-121-19A aredistinguished by the fact that their 5' ends are located 14 amino acidscloser to the carboxy terminal of NS5a than the previously observedstart of clones Y5-10, Y5-12, and Y5-26. None of the above clones hasits 3' end interior to that observed in the clone Y5-10. Thus a minimalsequence of this epitope is contained within amino acid sequence (SEQ IDNO:272).

B. Antigenic Clones from the ENV Library.

The ENV library was screened with HGV serum J21094. This serum (J21094)was identified as HCV positive based on the first generation (c-100) HCVtest. Subsequent testing of the initial J21094 serum sample, and ofsubsequently obtained J21094 samples, by PCR and with other HCV antigensconfirmed that the source individual for the serum was HCV infected.Evidence for the presence of HGV nucleic acid was obtained via PCRanalysis using the 470-20-1 and NS5 primer sets.

A number of phage clones were identified as immunoreactive with J21094serum. The phage were plaque purified and sequenced. Seven of the clones(Q7-12-1, Q7-16-2-2, Q7-15-2, Q7-17-2-1, Q7-19-1, and Q7-19-2-1)contained the same insert. The nucleotide sequence for Q7-12-1 ispresented as SEQ ID NO:143 (polypeptide sequence, SEQ ID NO:144).

One additional clone, Q7-16-1, obtained by the method just described,has the same 5' end as Q7-12-1, but is 26 amino acids shorter at the 3'end.

C. Antigenic Clones from the NS3 Library.

A one to one mixture of the F9/R9 phage and F10/R10 phage were screenedusing the following sera: PNF 2161, J21689 and E57963. Both J21689 andE57963 are sera that test co-positive for HCV and HGV by PCR (usingmultiple primers). Each immunoscreening was of 10 plates orapproximately 150,000 phage. Some of the immunopositive clonesidentified in these screens are as follows.

Clone Y12-10-3 (polynucleotide sequence, SEQ ID NO:145; polypeptidesequence, SEQ ID NO:146) was identified by its immunoreactivity withJ21689 serum. The clone expresses an 88 amino acid insert from HGV NS3.

Clone Y12-15-1 (polynucleotide sequence, SEQ ID NO:147; polypeptidesequence, SEQ ID NO:148) was identified by its immunoreactivity withE57963 serum. The clone expresses a 64 amino acid insert from the NS3protein of HGV. This sequence is located approximately 70 amino acids 5'to clone Y12-10-3.

D. Antigenic Clones from the NS2 Library.

Multiple positive plaques were isolated by screening the NS2 librarywith HGV-positive serum T56633. Eleven of these plaques weresubsequently purified and DNA sequenced. The locations of the insertscontained within these plaques (relative to SEQ ID NO:14) are delineatedin Table 20.

                  TABLE 20                                                        ______________________________________                                        CLONE     HGV VAR   SERA      START* STOP                                     ______________________________________                                        Q9-18-5   PNF       T56633    3071   2778                                     Q9-18-3   PNF       T56633    2951   2745                                     Q9-20-4   PNF       T56633    3002   2745                                     Q9-18-2   PNF       T56633    2990   2745                                     Q9-20-8   PNF       T56633    3062   2745                                     Q9-20-5   PNF       T56633    2972   2787                                     Q9-17-1   PNF       T56633    2990   2745                                     Q9-19-3   PNF       T56633    2982   2745                                     Q9-19-1   PNF       T56633    2982   2745                                     Q9-19-5   PNF       T56633    2984   2745                                     Q9-20-2   PNF       T56633    3027   2745                                     ______________________________________                                         *in this table the locations are given with respect to SEQ ID NO:14. The      actual sequence of the clones are the complement of the indicated             fragment.                                                                

All of the immunoclones express portions of the same open reading frame(ORF). This reading frame is encoded by the HGV polynucleotide strandthat is complementary to the sequence encoding the polyprotein. This ORFextends between nts 6322 and 6865 of the sequence complementary to SEQID NO:14. There is a Methionine that could serve as a site oftranslation initiation located at nt 6388 of the complementary strandthat would allow for the production of a 159 amino acid protein.

The smallest amino acid sequence common to all of the 11 sequencedclones is located between nts 6342 to 6606 (relative to thecomplementary strand of SEQ ID NO:14). The amino acid sequence encodedby this region of the negative strand of HGV-PNF 2161 is presented asSEQ ID NO:273.

The subcloning and subsequent Western blot analysis of immunoreactivenegative strand regions is described below.

E. Antigenic Clones from the VNS5A Library.

Approximately 1.5×10⁵ phage from the VNS5a library was plated out andsubsequently screened with the HGV-positive serum J29374 using theprocedures described in Example 13. Immunoscreening of the VNS5a librarywith J29374 resulted in the isolation of multiple positive plaques. Sixof these plaques were purified and subsequently DNA sequenced. Theoriginal strain of the DNA sequence obtained could be determined bywhich of the SISPA linker sequences was present at the 5' and 3' ends ofthe clones. The locations of the starts and stops of the obtained clones(relative to SEQ ID NO:14) and their source sera are summarized in Table21.

                  TABLE 21                                                        ______________________________________                                                  HGV Variant                                                         Clone     Source     Sera       Start*                                                                             Stop                                     ______________________________________                                        Q11-14-2  AH0591     J29374     6525 6749                                     Q11-16-1  E21-A      J29374     6432 6935                                     Q11-10-2  T56633     J29374     6579 6710                                     Q11-18-2  T56633     J29374     6579 6758                                     Q11-22-1  T56633     J29374     6576 6680                                     Q11-9-1   T56633     J29374     6531 6851                                     ______________________________________                                    

All of these clones contain the sequence of the clone Q11-22-1 in common(SEQ ID NO:274). This amino acid sequence is located immediately 5' tothe minimal sequence of the Y5-10 epitope. Thus it defines an additionalunique epitope in HGV NS5a (along with Y5-10 and Y5-5). Comparison ofthe observed amino acid sequence of these 3 HGV variants with thesequence of the PNF-2161 and JC isolates reveals few amino acidsubstitutions.

EXAMPLE 14 Further Characterization of Immunoreactive Clones

A. Subcloning.

1. Y5 CLONES.

Clones Y5-10, Y5-16, and Y5-5 were selected for subcloning into theexpression vector pGEX-HisB. PCR primers were designed which removed theextraneous linker sequences at the end of these clones. These primersalso introduced (i) a NcoI site at the 5' end (relative to the codingsequence) of each insert, and (ii) a BamHI site at the 3' end of eachinsert. Using these primers (see Table 22), the DNA fragments wereamplified from 2 μl of the plaque pure stocks.

                  TABLE 22                                                        ______________________________________                                        Clone        Primer Set                                                       ______________________________________                                        Y5-10        Y5-10-F1     SEQ ID NO:99                                                     Y5-10-R1     SEQ ID NO:100                                       Y5-16        Y5-16F1      SEQ ID NO:101                                                    470ep-R3     SEQ ID NO:102                                       Y5-5         Y5-5-F1      SEQ ID NO:103                                                    470ep-R3     SEQ ID NO:102                                       ______________________________________                                    

Amplifications were performed as follows: 30 cycles of 94° C. for 1minute, 50° C. for 1.5 minutes, and 72° C. for 2 minutes. Afteramplification the resulting DNAs were purified using "WIZARD PCR," spincolumns, the samples eluted in 50 μl, and digested overnight with NcoIand BamHI. A minimum of 30 units of each enzyme was used in therestriction endonuclease digestions (NcoI, Boehringer Mannhiem; BamHI,Promega).

The digested PCR fragments were ligated overnight to expression vectorpGEX-HisB that had been digested with NcoI and BamHI. Each set ofligated plasmids was independently used to transform E. coli strainW3110, using a heat shock protocol (Ausubel, et al.; Maniatis, et al.).Transformants were selected on LB plates containing 100 μg/ml ampicillinand resistant colonies were used to inoculate 2 mls of LB containing 100μg/ml ampicillin. Cultures expressing non-recombinant sj26/his proteinwere also prepared.

After incubation overnight at 37° C. the cultures were diluted 1/10 into2 mls of fresh LB plus ampicillin and grown for an additional 1 hour at37° C. IPTG was added to a final concentration of 0.2 mM and thecultures were grown for an additional 3 hours at 37° C. The bacteriawere pelleted by centrifugation and the bacterial pellet was resuspendedin 100 μl PBS. To the pellet, 100 μl of 2× SDS sample buffer (0.125MTris, pH 6.8, 10% glycine, 5% β-mercaptoethanol, 2.3% SDS) was added.The resulting lysates were vortexed and heated to 100° C. for 5 minutes.Aliquots (15 μl) of each lysate were loaded onto a 12% acrylamideSDS-PAGE gel.

The expressed proteins were size-fractionated by electrophoresis. Theseparated proteins were transferred from the gel to nitrocellulosefilters using standard techniques (Harlow, et al.). An additional gelcontaining the expressed proteins was stained using coomasie blueprotein stain.

Transformants carrying plasmids Y5-10, Y5-5 and Y5-16 expressedsignificant amounts of correctly sized recombinant fusion proteins. Theidentity of the recombinant fusions were confirmed by incubating aWestern blot (prepared above) with a murine monoclonal antibody that isspecifically immunoreactive with sj26 (Sierra BioSource, Gilroy,Calif.).

Additional confirmation that the picked colonies contained theappropriate insert was obtained as follows. A phage solution for eachcolony was prepared by inoculating 40 μl of TE solution with a toothpickcontaining a small amount of bacteria putatively expressing arecombinant clone had been inoculated. A 5 μl sample was taken from eachsolution and separately PCR amplified.

The amplifications employed the appropriate forward primer, (e.g., Y5-10F for a colony putatively expressing Y5-10) and a reverse primer (SEQ IDNO:104) homologous to a sequence located 3' to the cloning sites of theplasmid pGEX-HisB. The PCR amplifications were for 25 cycles as follows:94° C. for 1 minute, 50° C. for 1.5 minutes and 72° C. for 2 minutes.All of the colonies selected for further analysis produced a correctlysized DNA band with no other obvious bands under these conditions.

The immunoreactivity of the antigens expressed from the Y5-10, Y5-16, &Y5-5 inserts (expressed as sj26-his fusion proteins) was determined asfollows. Aliquots (15 μl) of the crude lysates prepared above weresize-fractionated by SDS-PAGE using a 12% acrylamide gel. The proteinswere electro-blotted ("NOVEX MINICELL MINIBLOT II," San Diego, Calif.)onto nitrocellulose filters. The filters were then individuallyincubated with one of the following sera: JC, PNF 2161, and super normalserum 4 (SN4) (R05072) as a negative control. In addition, one filterwas incubated with anti-sj26 monoclonal antibodies (RM001; SierraBioSource).

As expected, the recombinant protein produced by the bacteria expressingthe antigens encoded by the Y5-10, Y5-5, and Y5-16 inserts all reactedwith JC sera. No reactivity was observed with either PNF 2161 or SN4sera. All proteins appeared to be expressed at similar levels asdetermined by their reactivity to the anti-sj26 monoclonal antibody. TheY5-5 and Y5-10 encoded proteins were selected for further purification.

E. coli carrying Y5-5- and Y5-10- containing pGEX-HisB vectors werecultured and expression of the fusion protein induced as describedabove. The cells were lysed in PBS, containing 2 mM PMSF, using a FrenchPress at 1500 psi. The crude lysate was spun to remove cellular debris.The supernatant was loaded onto the glutathione affinity column at ahigh flow rate and the column was washed with 10 column volumes of PBS.The Y5-5 and Y5-10 fusion proteins were eluted with 10 mM Tris pH 8.8containing 10 mM glutathione.

Each of the fusion protein samples was diluted 1/10 with Buffer A (10 mMTris pH 8.8, containing 8M urea) and loaded onto a nickelcharged-chelating "SEPHAROSE" fast flow column. Each column wasrepeatedly washed with Buffer A until no further contaminants wereeluted. The fusion proteins were eluted using a gradient of imidazole inbuffer A. An imidazole gradient was run from 0 to 0.5M imidazole in 20column volumes. Fractions were collected.

Each set of fractions was analyzed by standard SDS-PAGE using 12%polyacrylamide gels. Pools of the Y5-5 and Y5-10 fusionprotein-containing fractions were separately made.

FIGS. 8A to 8D show the results of Western blot analysis of thefollowing samples (μg/lane): lane 1, Y5-10 antigen 1.6 μg; lane 2, Y5-10antigen 0.8 μg; lane 3, Y5-10 antigen 0.4 μg; and lane 4, Y5-10 antigen0.2 μg. Human serum JC (FIG. 8A) and Super Normal 2 serum (FIG. 8B) werediluted 1:100. The anti-GST mouse monoclonal antibody RM001 (FIG. 8C)was diluted 1:1000. FIG. 8D shows the Y5-10 antigen resolved bySDS-PAGE, transferred onto the nitrocellulose membrane and stained withPonceau S protein stain (Kodak, Rochester, N.Y.; Sigma). Arrow indicatesthe location of Y5.10 antigen. These results demonstrate that Y5-10 isspecifically immunoreactive with N-(ABCDE) human serum JC.

FIGS. 9A to 9D show the results of Western blot analysis of thefollowing samples: lane 1, Y5-5 antigen 3.2 μg; lane 2, Y5-5 antigen 1.6μg; lane 3, Y5-5 antigen 0.8 μg; lane 4, Y5-5 antigen 0.4 μg; lane 5,Y5-5 antigen 0.2 μg; lane 6, GE3-2 antigen 0.4 μg; and lane 7, Y5-10antigen 0.4 μg. Human serum JC (FIG. 9A), T55806 (FIG. 9B), and SuperNormal 2 serum (FIG. 9C) were diluted 1:100. RM001, the anti-GST mousemonoclonal antibody, (FIG. 9D) was diluted 1:1000. Arrows indicate thelocations of antigens Y5.5, GE3.2 and Y5.10. These results show specificimmunoreactivity of the Y5-5 antigen with the JC serum. Further, theantigens GE3-2 and Y5-10 were reactive with T55806. However, the Y5-5antigen was not reactive with the HGV-positive sera T55806.

The Y5-10 antigen was also size-fractionated by SDS polyacrylamide gelelectrophoresis. The gel was stained using coomasie blue protein stain.The gel was scanned for purity with a laser densitometer. The purity ofthe Y5-10 fusion protein was approximately 95%.

2. ENV CLONES.

The immunoclone Q7-12-1 was originally isolated by screening the ENVepitope library with the HGV positive sera J21094. Sequence specificprimers were employed to isolate the HGV insert contained within theQ7-12-1 λgt11 clone. The Q7-12-1 insert was excised and cloned intopGEX-Nde. The sequence of the insert was confirmed by the DNA sequencing(SEQ ID NO:275).

3. NS3 CLONES.

The immunoclone Y12-15-1 was originally isolated by screening the NS3epitope library with the HGV positive sera E57963. Sequence specificprimers were employed to isolate the HGV insert contained within theY12-15-1 λgt11 clone. The Y12-15-1 insert was excised and cloned intopGEX-Nde. The sequence of the insert was confirmed by the DNA sequencing(SEQ ID NO:276).

The immunoclone Y12-10-3 was originally isolated by screening the NS3epitope library with the HGV positive sera J21689. Sequence specificprimers were employed to isolate the HGV insert contained within theY12-10-3 λgt11 clone. The Y12-10-3 insert was excised and cloned intopGEX-Nde. Production of fusion proteins by selected clone was evaluatedby Western blot analysis. The sequence of the insert was confirmed bythe DNA sequencing (SEQ ID NO:277).

4. NS2 CLONES.

Multiple negative strand immunoclones derived from sequencescomplementary to the sequences of the NS2 region of SEQ ID NO:14 wereisolated. There are at least 2 significant ORFs encoded by the negativestrand of HGV. The first of these ORFs, represented by the Q9 series ofclones was described above. The second of these ORFs is located betweennts 6723 and 7259 of the complement of SEQ ID NO:14 and also possess a5' methionine at nt 6774. The second ORF encodes a 162 amino acidprotein.

Selected portions of the sequences of both of these negative strand ORFswere cloned into the expression vector pGEX-Nde. All of these subcloneswere obtained by the PCR amplification of PNF 2161 SISPA material usingappropriate oligonucleotide primers, thus they contain the sequence ofthe HGV-PNF 2161 variant. Table 23 indicates the names, size of the ORFand locations relative to the complement of SEQ ID NO:14.

                  TABLE 23                                                        ______________________________________                                        NAME/ORF   ORF       FROM NT (ATG)                                                                              TO NT                                       ______________________________________                                        5' NEG ORF 159 AA    6388         6865                                        3' NEG ORF 162 AA    6722         7258                                        NORF-F1/R1 3'        7107         7259                                        NORF-F4/R1 3'        6900         7259                                        NORF-F4/KR2                                                                              3'        6901         7172                                        NORF-F2/R1 3'        6744         7259                                        NORF-KF2/R4                                                                              5'        6684         6865                                        NORF-KF1/R2                                                                              5'        6881         6742                                        NORF-F3/R2 5'        6389         6742                                        NORF-F2/R3 3'        6744         6899                                        K3P-KF2/KR1                                                                              5'        6684         6772                                                   3'        6744         6791                                        ______________________________________                                    

The first 2 lines of this table identify the locations of the NS2 region5' and 3' negative strand ORFs relative to the complement of SEQ IDNO:14. The remaining lines indicate the specific nucleotide sequencesexpressed by all of the 9 clones. Note that several of the clonesexpress amino acids located 5' to the hypothetical HGV initiatingmethionine of the ORF. Also note that the last clone listed,K3p-KF2/KR1, is a chimera expressing the indicated portions of the 5'ORF followed by the indicated portions of the 3' ORF.

All of the DNA fragments were subsequently cloned into pGEX-Nde. Insertcontaining clones were also identified and confirmed.

5. NS5A CLONES.

Table 24 lists a number of NS5a clones and the regions of SEQ ID NO:14to which they correspond.

                  TABLE 24                                                        ______________________________________                                                    HGV                                                               Name        Source       Start  Stop                                          ______________________________________                                        EXY10-F2    PNF          6416   6827                                          EXY10-F3    PNF          6537   6827                                          Q11-F1-R1   T56633       6537   6680                                          Q11-F1-R2   T56633       6537   6827                                          Q11-F2-R1   T56633       6576   6680                                          Q11-F2-R2   T56633       6576   6827                                          Y5-12       PNF          6633   6917                                          EXY12       PNF          6918   6977                                          EXY10F14    PNF          6822   6977                                          ______________________________________                                    

These sequences were cloned into the vector pGEX-Nde for expression ofthe encoded protein antigens.

B. Western Blot Analysis of Selected HGV Subclones.

To determine the reactivity of both the negative and positive strandconstructs described above whole cell lysates from bacteria expressingthe various HGV subclones were prepared essentially as described inExample 13B. Aliquots of the expressed proteins were then fractionatedby SDS-PAGE, the proteins transferred to nitrocellulose filters, and thefilters probed with HGV-positive or control sera (e.g., anti-SJ26 MABRM01). The blots were incubated with an appropriate reporter antibody.

With respect to the HGV proteins tested, clear immunoreactivity to theprotein NORF-F3/R2 was detected with the HGV sera J21689 and T56633. TheNORF-F3/R2 subclone expresses the amino acid sequences that were alsoencoded by the Q9 series of negative strand epitope clones. The observedstrong reactivity with HGV sera T56633 confirms the immunoreactivity ofthis region of the negative strand of HGV. Reactivity to the NORF-F3/R2protein was not observed with the sera from the HGV negative individualR04316 or any of 5 other HGV negative supernormal sera tested.

Additional blots indicated that the other major 5' ORF clone NORFKF2-R4, which expresses amino acids of the carboxy terminal half of the5' negative strand ORF located does not react with the HGV-positive seraT56633. This observation in conjunction with the locations of the Q9epitope clones described above suggest that the immunogenic epitope ofthis portion of the negative strand is contained within the 55 aminoacid delineated above (SEQ ID NO:273). The fact that this sequence isrecognized by other HGV antisera, including J21689, indicates thatimmunoreactivity towards this sequence is relatively widespread amongHGV infected individuals.

Further, clear immunoreactivity with the Y12-10-3 protein was observedwith the HGV-infected sera J21689, J29374, and E57963. The specificityof this reactivity is additionally supported by the failure to observeimmunoreactivity with the HGV antisera J29374 or E57963 in the absenceof the induction of Y12-10-3 protein expression by IPTG. No reactivityto Y12-10-3 was observed with any of 7 supernormal sera tested.

EXAMPLE 15 A Multi-Antigen HGV Diagnostic Assay

Although the epitope clones described above do not appear to be reactivewith all HGV PCR-positive sera, many of these clones react with asubstantial fraction of the HGV infected sera they have been testedagainst. Additionally these proteins have not exhibited substantialcross reactivity with HGV-negative sera. It is therefore possible toconstruct a diagnostic assay in which several of these proteins arecombined so that the individual reactivities of the protein are summed.Such an assay is expected to have a relatively high sensitivity for thedetection of HGV-positive sera and a relatively low backgroundreactivity with HGV-negative sera.

Exemplary epitopes/antigens useful in such an assay include, but are notlimited to, NORF-F3/R2 (NS2-Neg strand), Y12-10-3 (NS3), Q11-F2-R1(NS5a), Y5-10 (NS5a), Y5-5 (NS5a), Q11-F2-R2 (combines 2 epitopes ofNS5a).

For this assay, individual antigens are typically selected that containdifferent unique epitopes that recognized different subset ofHGV-positive sera. Further, such antigens typically do not significantlyreact with HGV-negative sera. Following the guidance of the presentinvention, additional useful immunogenic clones can be isolated.

A multi-antigen diagnostic assay can take many formats. In oneembodiment, the assay might entail immobilizing each of, et al., 5 HGVproteins and control proteins at separate locations on a nitrocellulosestrip or other convenient solid phase format. Alternatively thenon-viral portions of, for example, an HGV-fusion protein could bemodified, either by insertions or deletions such that they wouldnaturally migrate to easily distinguishable locations upon SDS PAGE andsubsequent Western blot analysis. Strips are then incubated in testsera. After detection of bound antibody, a serum may then be scoredbased on (i) the number of antigens with which it is immunoreactive, and(ii) the strength of the immunological reactions. Reactivity to anon-HGV control protein would render a serum un-typeable. Reactivitywith no HGV protein would classify a serum as HGV-negative.

ELISA-based screening assay can be formed by combining purified antigenproteins in a single reaction zone or by creating protein constructsthat express 2 or more of the reactive epitopes as a single protein(e.g., a HGV mosaic polypeptide). The methods to construct mosaicpolypeptides is described herein. Q11-F2-R2 construct described above,in fact, represents a "matrix protein" that encodes 2 individualepitopes in a single polypeptide chain. Western blot assays may serve asa confirmatory assay for such an ELISA screening test.

Alternatively or in addition, full length HGV proteins, such as E2, NS5aand NS3 might be placed in a single reaction zone. Sera reactive withsuch proteins may also be confirmed as HGV positive by Western blotassay.

EXAMPLE 16 Expression of Large HGV Polypeptides

A. Expression of Larger HGV Antigens in E. coli

1. Cloning and Expression.

To identify conformational HGV epitopes (not covered by smalloverlapping HGV constructs or by phage library screening) larger HGVprotein constructs were generated in the pET-21a(+) vector (Novagen,Wis.) based on the prediction of cleavage sites (Bazan, et al., 1989;Chambers, et al., 1990b; Grakoui, et al., 1993; Kyte and Doolittle,1982). Individual HGV protein constructs were generated in a similarfashion to HGV sequences cloned into pGEX vectors.

Briefly, selected HGV sequences were RT-PCR amplified from a HGV(+)human sera source using HGV sequence specific primers. The primers wereengineered to contain appropriate restriction sites for cloningmanipulations in the pET vector. Coding sequences of interest weretypically inserted between the EcoRI site and the HindIII sites in thevector to produce 5' in-frame fusions with T7.Tag leader sequence and 3'in-frame fusion with a hexamer histidine sequence. T7.Tag (an 11 aminoacid sequence) allows the detection of the fusion proteins using ananti-T7.Tag monoclonal antibody (Novagen, Wis.). The histidine hexamerat the carboxyl end of the fusion protein allows the purification of theprotein using immobilized metal ion affinity chromatography.

HGV fragments were ligated into appropriately digested pET-21a(+)vectors. Ligated products were transformed into competent E.coli(HMS174; Novagen, Wis.). Plasmid DNA from transformed HMS174 wasanalyzed for the presence of HGV sequences by PCR, using primers T7F(SEQID NO:157) and T7R(SEQ ID NO:158), which are homologous to pET-21a(+)vector sequences flanking the inserted molecule. The size of the PCRproduct was the insert size plus approximately 260 bp derived from thevector.

For each construct the PCR results confirmed the presence of the insertsequences. Transformants with appropriate inserts were selected, plasmidDNAs with HGV inserts prepared and introduced into HMS174(DE3) competentE.coli (Novagen, Wis.) for the expression of HGV proteins.

Expression of HGV proteins was induced with 1 mM IPTG. Expression of theT7.Tag fusion proteins was monitored by the appearance of the predictedsize proteins on the Coomassie blue stained gel. Expression of thefusion proteins was confirmed by Western blot analysis using anti-T7.Tagantibody (Novagen, Wis.). HGV proteins expressed in pET-21a(+) vectorare shown in the Table 25. The start and end points of the expressedsequences are given relative to SEQ ID NO:14. The amino acid sequence ofGE-Cap is shown in SEQ ID NO:185.

                  TABLE 25                                                        ______________________________________                                                       Serum                                                          Name   Domain  Source  Start End   HGV aa                                                                              Size (KDa)                           ______________________________________                                        GE-Cap capsid  T55806   271*  480*  70   11                                   GE-E1a E1      PNF      594  1148  185   24                                   GE-E2  E2/NS1  PNF     1149  2183  345   41                                   GE-NS2b                                                                              NS2b    PNF     2904  3254  117   16                                   GE-N53 NS3     PNF     3255  5081  609   70                                   GE-NS4a                                                                              NS4a    PNF     5082  6083  334   40                                   GE-NS4b                                                                              NS4b    PNF     6084  6536  151   20                                   GE-NS4 NS4     PNF     5082  6536  485   57                                   GE-NS5a                                                                              NS5a    PNF     6537  7529  331   39                                   GE-NS5b                                                                              NS5b    PNF     7530  9044  505   59                                   ______________________________________                                         *These sequences are given relative to SEQ ID NO:178                     

FIG. 12 shows the expression of each HGV proteins demonstrated byWestern blot analysis with T7.Tag monoclonal antibody. The lanes in FIG.12 are as follows: Lane 1, pre-stained molecular weight marker(Bio-Rad); Lane 2, uninduced GE-Cap lysate; Lanes 3-11, IPTG inducedlysates of GE-Cap, E1a, E2, NS2b, NS3, NS4a, NS4b, NS4, and NS5b lysate,respectively. Lane 12 contained 1 μg of purified NS5a. Locations of eachantigen are marked with arrow heads. As shown in FIG. 12 all the HGVproteins were expressed in E.coli.

2. Western Blot Analyses of HGV proteins expressed in pET vector

Western blot analyses of the HGV protein expressed in pET vector wereperformed as described in Example 11C using E. coli whole cell lysatesand pre-absorbed sera. The results of these analyses demonstrated thatseveral of pET HGV proteins are specifically immunoreactive withHGV-positive human sera but not with HGV-negative human sera. GE-NS2b-1protein was immunoreactive with J21689 serum. The GE-NS5a-3 protein wasimmunoreactivity with several HGV (+) sera on Western blot analysis,including JC, T55806, T56633, J21689, E57963 and R0001. Among these seraT55806, J21689 and E57963 are HCV co-positive (by the PCR analysis).Neither GE-NS2b-1 nor GE-NS5a-3 were immunoreactive with several HGVnegative sera tested.

FIGS. 10A to 10F show the exemplary results of a series of Western blotexperiments examining the reactivity of antigens GE-NS2b and GE-NS5a3.The lanes in each blot of FIGS. 10A to 10F are as follows: Lane 1,uninduced GE-NS2b lysate; Lane 2, IPTG induced GE-NS2b lysate; Lane 3,uninduced GE-NS5a lysate; and Lane 4, IPTG induced GE-NS5a lysate. Eachblot was incubated with a human serum or mouse monoclonal antibody: FIG.10A, J29374; FIG. 10B, J21689; FIG. 10C, T56633; FIG. 10D, T43608 (supernormal serum); FIG. 10E, Anti-T7.Tag; and FIG. 10F, coomassie stainedgel. The serum or monoclonal antibody that was used is indicated aboveeach blot. Human sera were diluted 1:100 and anti-T7.Tag mousemonoclonal antibody was diluted 1:1000.

In addition to the sera listed above, additional HGV-PCR positive serahave been screened using GE-NS5a. The results of all these analyses havedemonstrated the reactivity of the GE-NS5a antigen with multipleHGV-infected sera.

GE-NS5b was immunoreactive with HGV(+) sera JC and T55806 but was notimmunoreactive with HGV(-) negative sera tested. FIGS. 13A to 13E showthe results of a series of Western blot experiments examining thereactivity of antigen GE-NS5b. The lanes in each blot the figures are asfollows: Lane 1, pre-stained molecular weight marker (Bio-Rad); Lane 2,uninduced GE-NS5b lysate; Lane 3, IPTG induced GE-NS5b lysate.

Each blot was incubated with a human serum or mouse monoclonal antibody:FIG. 13A, anti-T7.Tag monoclonal antibody; FIG. 13B, JC; FIG. 13C,T55806; and FIG. 13D, T43608 (super normal serum). FIG. 13E is aCoomassie Stain.

FIGS. 14A to 14D show the results of a series of Western blotexperiments examining the reactivity of antigen GE-E2. The lanes in eachof FIGS. 14A to 14D are as follows: Lane 1, pre-stained molecular weightmarker (Bio-Rad); Lane 2, uninduced GE-E2 lysate; Lane 3, IPTG inducedGE-E2 lysate. Each blot was incubated with a human serum or mousemonoclonal antibody: FIG. 14A, anti-T7.Tag monoclonal antibody; FIG.14B, 3831781; and FIG. 14C, T43608 (super normal serum). FIG. 14D isCoomassie Stain. The serum or monoclonal antibody that was used isindicated above each blot. GE-E2 protein was immunoreactive withHGV-positive serum 3831781 but was not immunoreactive with supernormalserum T43608 (FIGS. 14B and 14C, respectively).

Antigens GE-Cap and GE-NS4a were also specifically immunoreactive withHGV(+) serum J21689.

B. Expression larger HGV Antigens in Insect Cells.

Expression of proteins using recombinant baculoviruses offers thefollowing advantages (i) a high level of recombinant protein expression,and (ii) the benefits of a higher eucaryotic system, including efficientprotein translocation and modification. This system is particularlyuseful for expression of translocated proteins, e.g., HGV E1, E2 andNS2a.

1. Cloning and Expression.

Spodoptera frugiperda insect cell culture Sf21 and a derivative ofAutografa californica nuclear polyhedrosis virus "BACULOGOLD"(Pharmingen, San Diego, Calif.) were used for expression of HGVpolypeptides. Established protocols were used for insect cellcultivation and for generation of recombinant baculoviruses byco-transfection of baculovirus plasmid transfer vectors with linearizedbaculovirus DNA (King, 1992). Conventional techniques were used forconstruction of baculovirus plasmid transfer vectors (Maniatis, et al.;Sambrook, et al.).

The baculovirus transfer vector pAcYM1 (King, et al., 1992) was modifiedby ligating a double-stranded oligonucleotide coding for a Histidinehexamer into the vector's BamHI cloning site (vector designatedpAcYMIH). A stop codon (TAA) was placed after the Histidine hexamersequence. This provides a histidine hexamer on the carboxy-termini ofexpressed proteins. The BamHI cloning site of the pAcYMI parent vectorremained intact in the pAcYMIH and could be used for cloning variousgenes in-frame with the Histidine hexamer. The histidine hexamerprovides a method of rapid and efficient purification of the expressedprotein (Janknecht, et al., 1991).

A second baculovirus transfer vector, pVT-Bac, was also modified in asimilar manner to provide a histidine hexamer on the carboxy-termini ofexpressed proteins. pVT-Bac like the pAcYMI vector contains a stronglate polyhedrin promoter. In addition, pVT-Bac also provides a stronginsect translocation signal sequence to ensure efficient translocationof the expressed proteins (Tessier, et al., 1991). The pVT-Bac vectorwas modified by ligating a double-stranded oligonucleotide coding for ahistidine hexamer into the vector's BamHI cloning site (yielding thepVT-BacH vector). The BamHI cloning site of the pVT-Bac parent vectorremains intact in the obtained pVT-BacH vector and can be used forcloning genes in-frame with the insect leader sequence and the histidinehexamer sequence.

DNA fragments coding for various HGV genes were obtained by reversetranscription PCR. Regions of the HGV genome were selected according topredicted cleavage sites (Bazan, et al., 1989; Chambers, et al., 1990b;Grakoui, et al., 1993; Kyte and Doolittle, 1982). The following primerpairs were used in RT-PCR amplification reactions using PNF 2161 sourcenucleic acid: E1, SEQ ID NO:242, SEQ ID NO:243; E2B (HGV signalsequence), SEQ ID NO:246, SEQ ID NO:247; E2C (insect signal sequence),SEQ ID NO:244, SEQ ID NO:245; NS2a, SEQ ID NO:248, SEQ ID NO:249; NS2b,SEQ ID NO:250, SEQ ID NO:251; NS3, SEQ ID NO:252, SEQ ID NO:253; NS4a,SEQ ID NO:254, SEQ ID NO:255; NS4b, SEQ ID NO:256, SEQ ID NO:257; NS5a,SEQ ID NO:258, SEQ ID NO:259; NS5b, SEQ ID NO:260, SEQ ID NO:261; andE1-E2-NS2a, SEQ ID NO:262, SEQ ID NO:263.

Amplified DNA fragments were digested with BamHI or Bg1II endonucleasesand cloned into BamHI cut pAcYMI, pAcYMIH, pVT-Bac or pVT-BacH vectors.Sequences coding for the E1 and E2 carboxy-terminal anchors as well as ahydrophobic sequence at the carboxy-terminus of NS5b were deleted inorder to facilitate subsequent protein purification.

The recombinant baculovirus plasmid transfer vectors containing HGVsequences were co-transfected with linearized baculovirus DNA and therecombinant viruses were selected as white foci in presence of X-gal(King, et al., 1992). Recombinant viruses were twice plaque-purified andpropagated. Monolayers of Sf21 cells were infected with the recombinantbaculoviruses at the multiplicity 5 p.f.u. per cell and incubated at 27°C. for 60 h. The cells were washed with PBS and lysed in TNN buffer (50mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% "NONIDET-P40"). Inclusion bodieswere isolated by spinning the cell samples at 14k for 5 minutes. Theinclusion bodies were resuspended in protein dissociation buffer (10%2-mercaptoethanol, 10% SDS, 25% glycerol, 10 mM Tris-HCl pH 6.8, 0.02%Bromphenol blue) and incubated at 100° C. for 10 minutes.

The protein expression patterns analyzed by SDS-PAGE. Proteins wereseparated by 0.1% SDS-18% PAGE and stained with Coomassie brilliantblue. The majority of the HGV proteins were expressed to a high leveland could be easily detected on the Coomassie blue stained gels. NS5aand NS2a polypeptides were detected by ³⁵ S methionine protein labeling(King, et al., 1992).

HGV E2 protein glycosylation was examined as follows. Sf21 cells wereinfected with recombinant baculoviruses and processed as describedabove. Proteins were separated by 0.1% SDS-12% PAGE, electroblotted ontoan "IMMOBILON-P" (Millipore, Bedford Mass.) membrane and reacted withGalanthus nivalis agglutinin (Boehringer Mannheim DIG Glycandifferentiation kit) which is specific for mannose residues. The HGV E2protein that was expressed with its own signal sequence was extensivelyglycosylated, indicating that the predicted E2 signal sequence canfunction as such.

2. Immunofluorescence Assay Analysis.

SF21 insect cells were infected with the baculovirus-HGV constructsdescribed above. Cells were harvested, spun at 1.5K rpm for 3 minutes,washed in 1× PBS, and spun again.

For Immunofluorescence Assay (IFA) (King, et al., 1992) the cells wereresuspended in PBS and layered into the wells of glass slides such thatthe cells formed a sub-confluent layer in the wells of the slides. Theslides were air-dried. The cells fixed with pre-chilled -70° C. acetonefor 10 minutes and rehydrated with PBS for 5 minutes. The excess PBS wasremoved by blotting. The fixed cells were treated for one hour with thefollowing "Blocking" buffer: 40 mM Tris HCl pH 7.5, 3% goat serum, 1%BSA, 1% nonfat milk and 0.1% gelatin.

Primary antibody was then added to the fixed cells. Primary antibodiesincluded a series of human HGV-positive sera and a positive controlmonoclonal antibody. Before use, the sera were pre-absorbed fornon-specific proteins using insect cell lysate. Pre-absorption wascarried out overnight at 4° C. Uninfected SF21 cells were used as anegative control. After addition of a selected primary antibody (sera),the slides were incubated for 2 hours then washed several times with PBSand excess buffer removed. A secondary antibody conjugated withfluorescein (0.5 μg/ml conc.) was then added to the samples on theslides. The incubation time and temperature for the secondary antibodywas the same as for the primary antibody. After incubation, slides werewashed in PBS and mounted with a cover slip. The fluorescence of thecells was then determined using a fluorescence microscope.

The results of this analysis were as follows. Cells expressing HGVantigen E1-E2-NS2a were immunoreactive with 4/10 HGV-positive sera andweakly immunoreactive with an additional 2/10 sera. Cells expressing E1were weakly immunoreactive with 1/10 sera. Cells expressing E2 wereimmunoreactive with 3/10 sera and weakly immunoreactive with 1/10 sera.None of the cells carrying HGV antigens were immunoreactive withsupernormal control sera.

3. Western Blot Analyses of HGV proteins expressed in baculo vector

Western blot analyses of the HGV proteins expressed in recombinantbaculo virus infected Sf21 insect cells were also performed. Inclusionbodies were prepared as described above and subjected to Western blotanalysis. Western blot analysis was performed using pre-absorbed sera.The results of the analyses demonstrated that E2 proteins (one varianthaving the endogenous HGV signal sequence, E2B, and a second variantcarrying an insect signal sequence, E2C) were specificallyimmunoreactive with HGV(+) serum 3831781.

FIGS. 15A to 15D show the results of a series of Western blotexperiments examining the reactivity of baculo antigens E2B and E2C. Thelanes in each blot of FIGS. 15A to 15D were as follows: Lane 1,pre-stained molecular weight marker (Bio-Rad); Lane 2, E2B lysate; Lane3, E2C lysate; Lane 4, β-galactosidase lysate. Each blot was incubatedwith a human or rabbit serum : FIG. 15A, rabbit anti-E2 antibody; FIG.15B, 3831781 (an HGV-PCR-positive serum); FIG. 15C, 3838857 (anHGV-negative serum). FIG. 15D a Coomassie Stain. The serum or rabbitantibody that was used is indicated above each blot. Human sera werediluted 1:100 and rabbit serum was diluted 1:1000.

Further, HGV antigen NS2b protein expressed in insect cells wasimmunoreactive with J21689. These results are consistent with theresults obtained with pET expressed HGV proteins.

C. Expression of Larger Antigens in Vaccinia.

1. Cloning and Expression.

Various regions of HGV genome were integrated into vaccinia virus genomefor expression. An exemplary HGV polypeptide expression strategy isgiven in FIG. 16. HGV (PNF 2161 variant) proteins expressed in vacciniavirus are schematically illustrated in FIG. 16. Full length polyproteinis drawn (not to scale) by an open box indicating regions of predictedproteins: C=highly basic protein, 4A=NS4A, 4B=NS4B, 5a=NS5A, 5b=NS5B.The individual boxes with nucleotide locations (below the polyprotein)represents exemplary regions of HGV for expression in vaccinia virus.The number in the box stands for recombinant virus nomenclature. Virus#1 was derived from the highly basic protein region of HGV Stain T55806(SEQ ID NO:185).

Two sets of recombinant viruses were generated. The first set containedHGV sequences that correspond to individual protein domains based onsequence analysis of HGV cDNA (FIG. 16, fragments #1 to #9). The secondset contained HGV sequences that spanned multiple protein domains, up tofull length of HGV genome (FIG. 16, #10, #11, #14).

The various regions of the HGV genome were cloned into the multicloningsite of the vaccinia expression vector. A recombinant vaccinia virusexpression system was used that included bacterial phage T7 system andE. coli lac repressor for high level inducible expression (Fuerst, 1986;Elroy-Stein, 1989; Alexander, 1992; Moss, et al.). Therefore,recombinant protein is expressed only in the presence of an inducer,such as isopropyl beta-D-thiogalactoside (IPTG). Both direct cloning andPCR were used for plasmid construction. In the latter, restrictionendonuclease sites suitable for cloning into the vaccinia vector wereincorporated into primers used to amplify individual DNA fragment.

A polyhistidine tag was also incorporated into every clone coveringindividual domains of HGV for use in purifying the expressed proteins.HGV-PCR amplification products were digested with the appropriaterestriction enzymes and ligated into the vaccinia vector. Target HGVcDNA fragments were integrated into vaccinia virus genome throughhomologous recombination and drug (mycophenolic acid) selection(Falkner, 1988, Earl, 1991). Recombinant virus were plaque purified 4times before a viral stock was generated.

The length of each clone in nucleotides is indicated in FIG. 16. Thegroup of smaller clones (#1 to #9) are useful for HGV epitope mapping.The larger clones (e.g., #10, #11 and #14) are also useful for mappingthe HGV polyprotein cleavage sites experimentally. In addition to theclones shown in FIG. 16, additional recombinant viruses coveringmultiple domains from NS3 to NS5b can be constructed.

Expression plasmids were transfected into mammalian cells which had beeninfected with a parent vaccinia virus. CV-1 and BS-C-1 cells weremaintained in Minimum Essential Medium (MEM) supplemented with 10% fetalbovine serum. The cells were used for transfection (CV-1) andrecombinant virus selection and propagation (BS-C-1).

2. Evaluation of recombinant protein expression.

BS-C-1 cells were infected with recombinant virus in the presence orabsence of IPTG for 7 hours after which cells were labeled with ³⁵S-methionine for another one hour (Zhang, 1991). Briefly, 1×10⁶ BS-C-1cells were infected with recombinant virus at a multiplicity ofinfection (MOI) of 10 plaque forming unit (PFU) per cell for 1 h andthen supplemented with medium in the presence or absence of 5 mM IPTGfor another 6 h. Cells were pulse-labeled with 600 ul Methionine-freemedium supplemented with 2.5% dialyzed fetal bovine serum plus 60 uCi35S-methionine ("TRAN ³⁵ S-LABEL", ICN, Costa Mesa, Calif.) in thepresence or absence of 5 mM IPTG for another 60 min. Labeled cells werethen lysed on ice for 10 min in the presence of 100 mM Tris pH8.0, 150mM NaCl, and 1% "TRITON X-100." Nuclei were spun down and supernatantwas collected for analysis.

Cell lysate was analyzed by SDS-polyacrylamide gel electrophoresis(Fling, 1986; Schagger, 1987). Gels were fixed with 50% methanol and 10%acetic acid before they were treated with a fluorograph solution"AMPLIFY" (Amersham, Arlington Heights, Ill.). Gels were dried andexposed to X-ray film.

Using this method, expression of HGV polypeptides by viruses containinginserts #4 to #11, and #14 (FIG. 16) has been confirmed. Expression ofpolypeptides corresponding to other regions is confirmed in a similarmanner. For example, in a NS5a construct, upon induction by IPTG, aunique polypeptide was produced that migrated just below a 46 KDaprotein standard. This protein was not seen in the infection in theabsence of IPTG induction, establishing the identity of the protein asNS5a recombinant protein.

Further, limited immunoprecipitations using HGV region-specific antisera(for example, rabbit anti-sera raised against an isolated HGVpolypeptide from the region of interest) against ³⁵ S-Met labeled celllysate from individual virus infections was carried out to evaluate theprotein expression from recombinant viruses. For example, expression ofNS2, NS3, NS4B, NS5A and NS5B has been confirmed. An alternative method,to evaluate recombinant protein expression is to perform western blotanalysis with HGV-region-specific antisera.

When the full length HGV polyprotein was expressed in #14 virus (FIG.16), processed products of NS2, NS3 and NS5A were detected usingimmunoprecipitation with HGV region-specific antisera, demonstrating theusefulness of the full length HGV clone to evaluate polyproteinprocessing.

Using an expression strategy similar to that shown in FIG. 16, candidateHGV proteins/antigens can be expressed in yeast or CHO cells. Yeastoffers high level of expression, economical operation, and ease ofscaling up for commercial production. CHO cell lines allow secretion ofthe recombinant proteins into growth media for large scale proteinproduction and purification useful, for example, for vaccinedevelopment.

EXAMPLE 17 HGV Encoded Highly Basic Proteins

A. Determination of the Methionine used for Initiation IN THETRANSLATION OF HGV FROM PNF AND T55806.

The methionine located at nucleotide (nt) 459 (relative to SEQ ID NO:14)in the HGV-PNF 2161 variant is in-frame with the polyprotein. The"capsid" region appears to be 32 amino acid long. In other HGV isolates,such as T55806, this region is longer (e.g., about 83 amino acids). Themethionine located at nt 349 (relative to SEQ ID NO:14) in HGV-PNF 2161variant is not in-frame with the polyprotein sequence, but a methionineat the same position in HGV-T55806 variant is in frame with thepolyprotein. To see if there is a read-through or a ribosomal frameshift at this position in HGV-PNF 2161, the following experiments werecarried out.

Constructs were made containing (i) HGV genomic sequences having all theMET codons upstream of the HGV E1 region (e.g., in HGV-PNF 2161 thereare six such METs and five such in T55806), (ii) two different 3' endsfor each construct to allow determination of whether a ribosome shift ofread-through occurs. For a given genomic DNA, if both translatedproducts are the same size, that suggests they are terminatedprematurely at the stop codon. On the other hand, if read-through orframeshift occurs two products that differ by 55 amino acids areexpected.

A total of 21 constructs containing sequences from variants HGV-PNF 2161and HGV-T55806 were subcloned in a pGEX vector and correspondingproteins expressed in E. coli. Sizes of the resulting translationproducts were determined by both Coomassie stained gels and Westernsthat were blotted with monoclonal anti-GST antibody. Induced andun-induced samples were prepared for each construct.

The results demonstrated that the size of the protein productscorresponded to that expected by translation initiating at the first METin-frame with the polyprotein. There was no evidence of frame-shiftingor read-through.

B. Alternative Encoded Highly Basic Proteins.

The method of Fickett (1982) was used to scan the genomic sequencesHGV-PNF 2161 and HGV-JC for sequences that potentially encode proteins(i) alternative to the previously described polyprotein, (ii) showingconservation between HGV-PNF 2161 and HGV-JC, and (iii) having predictedisoelectric points in excess of pH 10. Two such potential proteins wereidentified.

The first protein is encoded by residues 628 through 882 (relative toSEQ ID NO:14) in HGV-PNF 2161 and by residues 556 through 810 (relativeto SEQ ID NO:182) in HGV-JC. This protein is 85 amino acids long, isgreater than 75% homologous between HFV94-1 and JC9B, and has apredicted pI of 11.6-12.3.

The second protein is encoded by residues 6844 through 7125 in HGV-PNF2161 (relative to SEQ ID NO:14) and by 6772 to 7053 in HGV-JC (relativeto SEQ ID NO:182). This protein is 94 amino acids long, is greater than88% homologous between HGV-PNF 2161 and HGV-JC, and has a predicted pIof 12.4-12.7.

These exemplary two proteins represent potentially expressed highlybasic proteins of HGV.

EXAMPLE 18 Cloning Further HGV Isolates and Design of Diagnostic Primers

A. Construction of a cDNA Clone of HGV-PNF 2161.

A cDNA clone of the nearly full-length HGV genome from PNF 2161 wasconstructed by cloning three overlapping PCR products into the plasmidvector pGEM3Z (Promega, Madison, Wis.). The PCR products used in thisconstruction were obtained by reverse transcription with "SUPERSCRIPTII" (Gibco/BRL, Gaithersburg, Md.) followed by PCR using reactionconditions that allowed for the amplification of long target sequences("rTth-XL" polymerase and "XL PCR BUFFERS", Applied Biosystems, FosterCity, Calif.). The rTth enzyme used for these "long-range" PCR reactionshas proof-reading activity (i.e. 3' to 5' exonuclease activity) thatcorrects mis-incorporated nucleotides, thus providing for high fidelityPCR.

The three products used to construct the HGV genome included (i) aninternal 6.7 kb product (nt 2101 to 8834 of SEQ ID NO:14) amplifiedusing the primers GV75-36FE (SEQ ID NO:228) and GV75-7064RLE (SEQ IDNO:229), (ii) a 2.8 kb 5'-end product (nt 38 to 2899 of SEQ ID NO:14)amplified using 28F (SEQ ID NO:230) and FV94-2864R (SEQ ID NO:231), and(iii) a 2.9 kb 3'-end product (nt 6449 to 9366 of SEQ ID NO:14)amplified using FV94-6439F (SEQ ID NO:232) and FV94-9331R (SEQ IDNO:233).

Initially, the 6.7 kb internal fragment was cloned into the "TA-vector"pCRII to create the clone HGV7. Subsequently, a 6.1 kb KpnI/EcoRIfragment was removed from HGV7 and combined with the KpnI/XbaI digested2.8 kb 5'-end product (the primer 28F contains an artificial XbaI site)and cloned into XbaI/EcoRI digested pGEM3Z. This 8.8 kb clone, whichlacks about 0.6 kb of the 3' portion of the HGV genome, was designatedHGV-KEX-2. To construct the nearly full-length HGV genome, the 3'-endHGV product was digested with NheI and EcoRI (the primer FV94-9331Rcontains an artificial EcoRI site) and cloned into NheI/EcoRI digestedHGV-KEX-2 plasmid creating a cloned HGV-PNF2161 sequence of 9329 nt (nt38 to 9366 of SEQ ID NO:14) that is designated 3Z-HGV94-6. The completesequence of 3Z-HGV94-6 is presented as SEQ ID NO:234.

The clone 3Z-HGV94-6 may be used to generate in vitro-transcribedfull-length HGV RNA or portions thereof (e.g., using SP6 polymerase).The RNA molecules can be used to transfect human cell lines. Thisapproach could be used to map the various regions of the viral genome,study its replication, and understand the mechanisms of HGVpathogenicity in human cells (Rice, et al., 1989; Sumiyoshi, et al.,1992; Yoo, et al., 1995).

B. Cloning the JC Variant.

One milliliter of JC serum was spun at 40,000 rpms (Beckman, SpincoRotor 70.1Ti) for 2 hours. The resulting pellet was extracted using"TRIREAGENT" (MRC, Cincinnati, Ohio), resulting in the formation of 3phases. The upper phase contained RNA only. This phase was taken and RNArecovered by ethanol precipitation.

HGV cDNA molecules were generated from the JC sample by two methods. Thefirst method was amplification (RT-PCR) of the JC nucleic acid sampleusing specific and nested primers. The primer sequences were based onthe HGV sequence obtained from PNF 2161 serum. The criteria used toselect the primers were (i) regions having a high G/C content, and (ii)no repetitious sequences.

The second method used to generate HGV cDNA molecules was amplificationusing HGV (PNF 2161) specific primers followed by identification of HGVspecific sequences with ³² P-labelled oligonucleotide probes. Such DNAhybridizations were carried out essentially as described by Sambrook, etal. (1989). The PCR derived clones were either (i) cloned into the "TA"vector (Invitrogen, San Diego, Calif.) and sequenced with vector primers(TAR and TAF), or (ii) sequenced directly after PCR amplification. Boththe probe and primer sequences were based on the HGV variant obtainedfrom the PNF 2161 serum.

These two approaches yielded multiply-overlapping HGV fragments from theJC serum. Each of these fragments were cloned and sequenced. Thesequences were aligned to obtain the HGV (JC-variant) consensus sequencepresented as SEQ ID NO:182 (polypeptide sequence, SEQ ID NO:183). Thesequence of each region of the HGV (JC-variant) virus was based on aconsensus from at least three different, overlapping, independentclones.

C. Other HGV Variants.

In addition to the HGV PNF 2161-variant and JC-variant sequences, threepartial HGV isolates have been obtained from the sera BG34, T55806 andEB20 by methods similar to those described above. The partial sequencesof these isolates are presented as SEQ ID NO:176 (BG34 nucleic acid),SEQ ID NO:177 (BG34 polypeptide), SEQ ID NO:178 (T55806 nucleic acid),SEQ ID NO:179 (T55806 polypeptide), SEQ ID NO:180 (EB20-2 nucleic acid)and SEQ ID NO:181 (EB20-2 polypeptide).

D. Alternative Primers for Diagnostic PCR.

PCR primers and corresponding assay development may be derived fromregions of the HGV genome(s) typically based on the analysis ofconserved regions. Based on comparisons of the HGV-JC variant and theHGV-PNF 2161 variant, the 5' untranslated region of HGV was selected asone such region for development of a further PCR-based diagnostic testfor the detection of HGV isolates. Two exemplary primers are FV-94-22F(SEQ ID NO:124) and FV94-724R (SEQ ID NO:125). These primers amplify anapproximately 728 bp fragment of the HGV genome.

Sequence analysis was performed on amplification products from reactionsemploying these two primers for 36 isolates of HGV (including PNF 2161and JC, see Table 26). An approximately 400 bp region (nt 69 to 469 ofSEQ ID NO:14) of the approximately 728 bp amplification product was usedfor multiple sequence alignments (Table 26) and further determination ofconserved regions (see below).

                  TABLE 26                                                        ______________________________________                                        SEQ ID  Serum                                                                 NO:     Code        Country  % ID PNF 2161                                    ______________________________________                                        186     S59         England  96.8                                             187     S368        England  98.8                                             188     S309        England  95.5                                             189     FZ          Australia                                                                              96                                               190     G21         Greece   97.8                                             191     G23         Greece   94.3                                             192     G59         Greece   93.6                                             193     E36         Egypt    94                                               194     R38730      USA      94.8                                             195     G281        Greece   97.8                                             196     G157        Greece   94.3                                             197     G154        Greece   96                                               198     G213        Greece   94.8                                             199     G204        Greece   98.3                                             200     G191        Greece   94.8                                             201     G299        Greece   94.8                                             202     T56957      USA      95.3                                             203     C01698      USA      98.8                                             204     T27034      USA      93.5                                             205     E57963      USA      98.5                                             206     R37166      USA      97.5                                             207     B5          Germany  95.5                                             208     B33         Germany  95.5                                             209     FH010       Australia                                                                              95                                               210     PNF2161     USA      100                                              211     JC          USA      96.3                                             212     7155        Peru     89.8                                             213     7244        Peru     89                                               214     K27         Korea    89.5                                             215     K30         Korea    89.5                                             216     T55875      USA      97.3                                             217     T56633      USA      93.5                                             218     EB20        Egypt    94.1                                             219     T55806      USA      95.6                                             220     BG34        Greece   94.8                                             221     BE12        Egypt    95                                               ______________________________________                                    

The development of an amplification-based (e.g., PCR) or probe-basedmethod/assay for the detection of HGV isolates in samples involves theselection of appropriate primer/probe sequences. Two criteria for suchan assay are low copy sensitivity and specificity for HGV sequences.Alignments of sequences (such as just described) can help guideprimer/probe selection and design.

Several criteria for selecting primers are as follows: (i) forward andreverse primers of a pair should not be significantly complementary insequence, and (ii) primers should not have significant selfcomplementarity or the potential to form secondary structures. Theseprecautions minimize the potential for generation of primer dimers oroligomers.

Primers may optimally be designed from sequence regions showing novariation among different isolates but may also be designed from regionsof less homology by incorporating mixed base synthesis or neutral bases,such as inosine, at those positions to account for known isolatedivergence. The following two groups of primers are examples of primersmay be employed in development of a PCR-based assay for detection of HGVgenomes: forward primers SEQ ID NO:222, SEQ ID NO:223 and SEQ ID NO:224;and reverse primers SEQ ID NO:225, SEQ ID NO:226 and SEQ ID NO:227.

Various combinations of primers, may be employed in development of anHGV diagnostic assay. Optimal combinations of primers are experimentallydetermined and typically address considerations for assay sensitivityand specificity. Such considerations include the following: (i) a PCRproduct length of 100-300 bp for efficient amplification and ease ofproduct detection; (ii) an ability to reproducibly detect at least 10copies of target HGV, and (iii) an ability to reproducibly detect amajority of HGV variants.

In addition, probe sequences may be similarly designed with mixed baseor neutral base syntheses and/or may be used at reduced stringency so asto detect a majority of HGV variants.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 277                                                (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SISPA primer, top strand Linker AB                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGAATTCGCGGCCGCTCG18                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Linker AB, bottom strand                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGAGCGGCCGCGAATTCCTT20                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 237 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PNF 2161 CLONE 470-20-1                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..237                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATTCGCGGCCGCTCGGGCTGTCTCGGACTCTTGGATGACCTCGAAT48                            GluPheAlaAlaAlaArgAlaValSerAspSerTrpMetThrSerAsn                              151015                                                                        GAGTCAGAGGACGGGGTATCCTCCTGCGAGGAGGACACCGGCGGGGTC96                            GluSerGluAspGlyValSerSerCysGluGluAspThrGlyGlyVal                              202530                                                                        TTCTCATCTGAGCTGCTCTCAGTAACCGAGATAAGTGCTGGCGATGGA144                           PheSerSerGluLeuLeuSerValThrGluIleSerAlaGlyAspGly                              354045                                                                        GTACGGGGGATGTCTTCTCCCCATACAGGCATCTCTCGGCTACTACCA192                           ValArgGlyMetSerSerProHisThrGlyIleSerArgLeuLeuPro                              505560                                                                        CAAAGAGAGGGTGTACTGCAGTCCTCCACGAGCGGCCGCGAATTC237                              GlnArgGluGlyValLeuGlnSerSerThrSerGlyArgGluPhe                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GluPheAlaAlaAlaArgAlaValSerAspSerTrpMetThrSerAsn                              151015                                                                        GluSerGluAspGlyValSerSerCysGluGluAspThrGlyGlyVal                              202530                                                                        PheSerSerGluLeuLeuSerValThrGluIleSerAlaGlyAspGly                              354045                                                                        ValArgGlyMetSerSerProHisThrGlyIleSerArgLeuLeuPro                              505560                                                                        GlnArgGluGlyValLeuGlnSerSerThrSerGlyArgGluPhe                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HAV-R1                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTTGACCAACTGAGTCTGAAGC22                                                      (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HAV-F1                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GATTGGAAATCTGATCCGTCCC22                                                      (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HCV- LANR                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCGCGACCCAACACTACTC19                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HCV 1532                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGGGGCGACACTCCACCA18                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470- 20-1-77F                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTCTTTGTGGTAGTAGCCGAGAGAT25                                                   (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470- 20-1-211R                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CGAATGAGTCAGAGGACGGGGTAT24                                                    (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer KL- 1                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCAGGATCCGAATTCGCATCTAGAGAT27                                                 (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer KL- 2                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ATCTCTAGATGCGAATTCGGATCCTGCGA29                                               (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: LAMBDA GT11, REVERSE PRIMER                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGCAGACATGGCCTGCCCGG20                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9392 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-PNF 2161 Variant                                  (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 459..9077                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACGTGGGGGAGTTGATCCCCCCCCCCCGGCACTGGGTGCAAGCCCCAGAAACCGACGCCT60                ATCTAAGTAGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGG120               GTGATGACAGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGT180               CTTAAGAGAAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGT240               GTTGGCCCTACCGGTGGGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGT300               TACCCACCTGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTG360               ACCAATAGGCGTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGAGAGGG420               ACTCCAAGTCCCGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACCCAGC473                      MetGlyProProSer                                                               15                                                                            TCCGCGGCGGCCTGCAGCCGGGGTAGCCCAAGAATCCTTCGGGTGAGG521                           SerAlaAlaAlaCysSerArgGlySerProArgIleLeuArgValArg                              101520                                                                        GCGGGTGGCATTTCCTTTTTCTATACCATCATGGCAGTCCTTCTGCTC569                           AlaGlyGlyIleSerPhePheTyrThrIleMetAlaValLeuLeuLeu                              253035                                                                        CTTCTCGTGGTTGAGGCCGGGGCCATTCTGGCCCCGGCCACCCACGCT617                           LeuLeuValValGluAlaGlyAlaIleLeuAlaProAlaThrHisAla                              404550                                                                        TGTCGAGCGAATGGGCAATATTTCCTCACAAATTGTTGTGCCCCGGAG665                           CysArgAlaAsnGlyGlnTyrPheLeuThrAsnCysCysAlaProGlu                              556065                                                                        GACATCGGGTTCTGCCTGGAGGGTGGATGCCTGGTGGCCCTGGGGTGC713                           AspIleGlyPheCysLeuGluGlyGlyCysLeuValAlaLeuGlyCys                              70758085                                                                      ACGATTTGCACTGACCAATGCTGGCCACTGTATCAGGCGGGTTTGGCT761                           ThrIleCysThrAspGlnCysTrpProLeuTyrGlnAlaGlyLeuAla                              9095100                                                                       GTGCGGCCTGGCAAGTCCGCGGCCCAACTGGTGGGGGAGCTGGGTAGC809                           ValArgProGlyLysSerAlaAlaGlnLeuValGlyGluLeuGlySer                              105110115                                                                     CTATACGGGCCCCTGTCGGTCTCGGCCTATGTGGCTGGGATCCTGGGC857                           LeuTyrGlyProLeuSerValSerAlaTyrValAlaGlyIleLeuGly                              120125130                                                                     CTGGGTGAGGTGTACTCGGGTGTCCTAACGGTGGGAGTCGCGTTGACG905                           LeuGlyGluValTyrSerGlyValLeuThrValGlyValAlaLeuThr                              135140145                                                                     CGCCGGGTCTACCCGGTGCCTAACCTGACGTGTGCAGTCGCGTGTGAG953                           ArgArgValTyrProValProAsnLeuThrCysAlaValAlaCysGlu                              150155160165                                                                  CTAAAGTGGGAAAGTGAGTTTTGGAGATGGACTGAACAGCTGGCCTCC1001                          LeuLysTrpGluSerGluPheTrpArgTrpThrGluGlnLeuAlaSer                              170175180                                                                     AACTACTGGATTCTGGAATACCTCTGGAAGGTCCCATTTGATTTCTGG1049                          AsnTyrTrpIleLeuGluTyrLeuTrpLysValProPheAspPheTrp                              185190195                                                                     AGAGGCGTGATAAGCCTGACCCCCTTGTTGGTTTGCGTGGCCGCATTG1097                          ArgGlyValIleSerLeuThrProLeuLeuValCysValAlaAlaLeu                              200205210                                                                     CTGCTGCTTGAGCAACGGATTGTCATGGTCTTCCTGTTGGTGACGATG1145                          LeuLeuLeuGluGlnArgIleValMetValPheLeuLeuValThrMet                              215220225                                                                     GCCGGGATGTCGCAAGGCGCCCCTGCCTCCGTTTTGGGGTCACGCCCC1193                          AlaGlyMetSerGlnGlyAlaProAlaSerValLeuGlySerArgPro                              230235240245                                                                  TTTGACTACGGGTTGACTTGGCAGACCTGCTCTTGCAGGGCCAACGGT1241                          PheAspTyrGlyLeuThrTrpGlnThrCysSerCysArgAlaAsnGly                              250255260                                                                     TCGCGTTTTTCGACTGGGGAGAAGGTGTGGGACCGTGGGAACGTTACG1289                          SerArgPheSerThrGlyGluLysValTrpAspArgGlyAsnValThr                              265270275                                                                     CTTCAGTGTGACTGCCCTAACGGCCCCTGGGTGTGGTTGCCAGCCTTT1337                          LeuGlnCysAspCysProAsnGlyProTrpValTrpLeuProAlaPhe                              280285290                                                                     TGCCAAGCAATCGGCTGGGGTGACCCCATCACTTATTGGAGCCACGGG1385                          CysGlnAlaIleGlyTrpGlyAspProIleThrTyrTrpSerHisGly                              295300305                                                                     CAAAATCAGTGGCCCCTTTCATGCCCCCAGTATGTCTATGGGTCTGCT1433                          GlnAsnGlnTrpProLeuSerCysProGlnTyrValTyrGlySerAla                              310315320325                                                                  ACAGTCACTTGCGTGTGGGGTTCCGCTTCTTGGTTTGCCTCCACCAGT1481                          ThrValThrCysValTrpGlySerAlaSerTrpPheAlaSerThrSer                              330335340                                                                     GGTCGCGACTCGAAGATAGATGTGTGGAGTTTAGTGCCAGTTGGCTCT1529                          GlyArgAspSerLysIleAspValTrpSerLeuValProValGlySer                              345350355                                                                     GCCACCTGCACCATAGCCGCACTTGGATCATCGGATCGCGACACGGTG1577                          AlaThrCysThrIleAlaAlaLeuGlySerSerAspArgAspThrVal                              360365370                                                                     CCTGGGCTCTCCGAGTGGGGAATCCCGTGCGTGACGTGTGTTCTGGAC1625                          ProGlyLeuSerGluTrpGlyIleProCysValThrCysValLeuAsp                              375380385                                                                     CGTCGGCCTGCCTCCTGCGGCACCTGTGTGAGGGACTGCTGGCCCGAG1673                          ArgArgProAlaSerCysGlyThrCysValArgAspCysTrpProGlu                              390395400405                                                                  ACCGGGTCGGTTAGGTTCCCATTCCATCGGTGCGGCGTGGGGCCTCGG1721                          ThrGlySerValArgPheProPheHisArgCysGlyValGlyProArg                              410415420                                                                     CTGACAAAGGACTTGGAAGCTGTGCCCTTCGTCAACAGGACAACTCCC1769                          LeuThrLysAspLeuGluAlaValProPheValAsnArgThrThrPro                              425430435                                                                     TTCACCATTAGGGGGCCCCTGGGCAACCAGGGCCGAGGCAACCCGGTG1817                          PheThrIleArgGlyProLeuGlyAsnGlnGlyArgGlyAsnProVal                              440445450                                                                     CGGTCGCCCTTGGGTTTTGGGTCCTACGCCATGACCAGGATCCGAGAT1865                          ArgSerProLeuGlyPheGlySerTyrAlaMetThrArgIleArgAsp                              455460465                                                                     ACCCTACATCTGGTGGAGTGTCCCACACCAGCCATTGAGCCTCCCACC1913                          ThrLeuHisLeuValGluCysProThrProAlaIleGluProProThr                              470475480485                                                                  GGGACGTTTGGGTTCTTCCCCGGGACGCCGCCTCTCAACAACTGCATG1961                          GlyThrPheGlyPhePheProGlyThrProProLeuAsnAsnCysMet                              490495500                                                                     CTCTTGGGCACGGAAGTGTCCGAGGCACTTGGGGGGGCTGGCCTCACG2009                          LeuLeuGlyThrGluValSerGluAlaLeuGlyGlyAlaGlyLeuThr                              505510515                                                                     GGGGGGTTCTATGAACCCCTGGTGCGCAGGTGTTCGAAGCTGATGGGA2057                          GlyGlyPheTyrGluProLeuValArgArgCysSerLysLeuMetGly                              520525530                                                                     AGCCGAAATCCGGTTTGTCCGGGGTTTGCATGGCTCTCTTCGGGCAGG2105                          SerArgAsnProValCysProGlyPheAlaTrpLeuSerSerGlyArg                              535540545                                                                     CCTGATGGGTTTATACATGTCCAGGGTCACTTGCAGGAGGTGGATGCA2153                          ProAspGlyPheIleHisValGlnGlyHisLeuGlnGluValAspAla                              550555560565                                                                  GGCAACTTCATCCCGCCCCCGCGCTGGTTGCTCTTGGACTTTGTATTT2201                          GlyAsnPheIleProProProArgTrpLeuLeuLeuAspPheValPhe                              570575580                                                                     GTCCTGTTATACCTGATGAAGCTGGCTGAGGCACGGTTGGTCCCGCTG2249                          ValLeuLeuTyrLeuMetLysLeuAlaGluAlaArgLeuValProLeu                              585590595                                                                     ATCTTGCTGCTGCTATGGTGGTGGGTGAACCAGCTGGCAGTCCTAGGG2297                          IleLeuLeuLeuLeuTrpTrpTrpValAsnGlnLeuAlaValLeuGly                              600605610                                                                     CTGCCGGCTGTGGAAGCCGCCGTGGCAGGTGAGGTCTTCGCGGGCCCT2345                          LeuProAlaValGluAlaAlaValAlaGlyGluValPheAlaGlyPro                              615620625                                                                     GCCCTGTCCTGGTGTCTGGGACTCCCGGTCGTCAGTATGATATTGGGT2393                          AlaLeuSerTrpCysLeuGlyLeuProValValSerMetIleLeuGly                              630635640645                                                                  TTGGCAAACCTGGTGCTGTACTTTAGATGGTTGGGACCCCAACGCCTG2441                          LeuAlaAsnLeuValLeuTyrPheArgTrpLeuGlyProGlnArgLeu                              650655660                                                                     ATGTTCCTCGTGTTGTGGAAGCTTGCTCGGGGAGCTTTCCCGCTGGCC2489                          MetPheLeuValLeuTrpLysLeuAlaArgGlyAlaPheProLeuAla                              665670675                                                                     CTCTTGATGGGGATTTCGGCGACCCGCGGGCGCACCTCAGTGCTCGGG2537                          LeuLeuMetGlyIleSerAlaThrArgGlyArgThrSerValLeuGly                              680685690                                                                     GCCGAGTTCTGCTTCGATGCTACATTCGAGGTGGACACTTCGGTGTTG2585                          AlaGluPheCysPheAspAlaThrPheGluValAspThrSerValLeu                              695700705                                                                     GGCTGGGTGGTGGCCAGTGTGGTAGCTTGGGCCATTGCGCTCCTGAGC2633                          GlyTrpValValAlaSerValValAlaTrpAlaIleAlaLeuLeuSer                              710715720725                                                                  TCGATGAGCGCAGGGGGGTGGAGGCACAAAGCCGTGATCTATAGGACG2681                          SerMetSerAlaGlyGlyTrpArgHisLysAlaValIleTyrArgThr                              730735740                                                                     TGGTGTAAGGGGTACCAGGCAATCCGTCAAAGGGTGGTGAGGAGCCCC2729                          TrpCysLysGlyTyrGlnAlaIleArgGlnArgValValArgSerPro                              745750755                                                                     CTCGGGGAGGGGCGGCCTGCCAAACCCCTGACCTTTGCCTGGTGCTTG2777                          LeuGlyGluGlyArgProAlaLysProLeuThrPheAlaTrpCysLeu                              760765770                                                                     GCCTCGTACATCTGGCCAGATGCTGTGATGATGGTGGTGGTTGCCTTG2825                          AlaSerTyrIleTrpProAspAlaValMetMetValValValAlaLeu                              775780785                                                                     GTCCTTCTCTTTGGCCTGTTCGACGCGTTGGATTGGGCCTTGGAGGAG2873                          ValLeuLeuPheGlyLeuPheAspAlaLeuAspTrpAlaLeuGluGlu                              790795800805                                                                  ATCTTGGTGTCCCGGCCCTCGTTGCGGCGTTTGGCTCGGGTGGTTGAG2921                          IleLeuValSerArgProSerLeuArgArgLeuAlaArgValValGlu                              810815820                                                                     TGCTGTGTGATGGCGGGTGAGAAGGCCACAACCGTCCGGCTGGTCTCC2969                          CysCysValMetAlaGlyGluLysAlaThrThrValArgLeuValSer                              825830835                                                                     AAGATGTGTGCGAGAGGAGCTTATTTGTTCGATCATATGGGCTCTTTT3017                          LysMetCysAlaArgGlyAlaTyrLeuPheAspHisMetGlySerPhe                              840845850                                                                     TCGCGTGCTGTCAAGGAGCGCCTGTTGGAATGGGACGCAGCTCTTGAA3065                          SerArgAlaValLysGluArgLeuLeuGluTrpAspAlaAlaLeuGlu                              855860865                                                                     CCTCTGTCATTCACTAGGACGGACTGTCGCATCATACGGGATGCCGCG3113                          ProLeuSerPheThrArgThrAspCysArgIleIleArgAspAlaAla                              870875880885                                                                  AGGACTTTGTCCTGCGGGCAGTGCGTCATGGGTTTACCCGTGGTTGCG3161                          ArgThrLeuSerCysGlyGlnCysValMetGlyLeuProValValAla                              890895900                                                                     CGCCGTGGTGATGAGGTTCTCATCGGCGTCTTCCAGGATGTGAATCAT3209                          ArgArgGlyAspGluValLeuIleGlyValPheGlnAspValAsnHis                              905910915                                                                     TTGCCTCCCGGGTTTGTTCCGACCGCGCCTGTTGTCATCCGACGGTGC3257                          LeuProProGlyPheValProThrAlaProValValIleArgArgCys                              920925930                                                                     GGAAAGGGCTTCTTGGGGGTCACAAAGGCTGCCTTGACAGGTCGGGAT3305                          GlyLysGlyPheLeuGlyValThrLysAlaAlaLeuThrGlyArgAsp                              935940945                                                                     CCTGACTTACATCCAGGGAACGTCATGGTGTTGGGGACGGCTACGTCG3353                          ProAspLeuHisProGlyAsnValMetValLeuGlyThrAlaThrSer                              950955960965                                                                  CGAAGCATGGGAACATGCTTGAACGGCCTGCTGTTCACGACCTTCCAT3401                          ArgSerMetGlyThrCysLeuAsnGlyLeuLeuPheThrThrPheHis                              970975980                                                                     GGGGCTTCATCCCGAACCATCGCCACACCCGTGGGGGCCCTTAATCCC3449                          GlyAlaSerSerArgThrIleAlaThrProValGlyAlaLeuAsnPro                              985990995                                                                     AGATGGTGGTCAGCCAGTGATGATGTCACGGTGTATCCACTCCCGGAT3497                          ArgTrpTrpSerAlaSerAspAspValThrValTyrProLeuProAsp                              100010051010                                                                  GGGGCTACTTCGTTAACACCTTGTACTTGCCAGGCTGAGTCCTGTTGG3545                          GlyAlaThrSerLeuThrProCysThrCysGlnAlaGluSerCysTrp                              101510201025                                                                  GTCATCAGATCCGACGGGGCCCTATGCCATGGCTTGAGCAAGGGGGAC3593                          ValIleArgSerAspGlyAlaLeuCysHisGlyLeuSerLysGlyAsp                              1030103510401045                                                              AAGGTGGAGCTGGATGTGGCCATGGAGGTCTCTGACTTCCGTGGCTCG3641                          LysValGluLeuAspValAlaMetGluValSerAspPheArgGlySer                              105010551060                                                                  TCTGGCTCACCGGTCCTATGTGACGAAGGGCACGCAGTAGGAATGCTC3689                          SerGlySerProValLeuCysAspGluGlyHisAlaValGlyMetLeu                              106510701075                                                                  GTGTCTGTGCTTCACTCCGGTGGTAGGGTCACCGCGGCACGGTTCACT3737                          ValSerValLeuHisSerGlyGlyArgValThrAlaAlaArgPheThr                              108010851090                                                                  AGGCCGTGGACCCAAGTGCCAACAGATGCCAAAACCACTACTGAACCC3785                          ArgProTrpThrGlnValProThrAspAlaLysThrThrThrGluPro                              109511001105                                                                  CCTCCGGTGCCGGCCAAAGGAGTTTTCAAAGAGGCCCCGTTGTTTATG3833                          ProProValProAlaLysGlyValPheLysGluAlaProLeuPheMet                              1110111511201125                                                              CCTACGGGAGCGGGAAAGAGCACTCGCGTCCCGTTGGAGTACGATAAC3881                          ProThrGlyAlaGlyLysSerThrArgValProLeuGluTyrAspAsn                              113011351140                                                                  ATGGGGCACAAGGTCTTAATCTTGAACCCCTCAGTGGCCACTGTGCGG3929                          MetGlyHisLysValLeuIleLeuAsnProSerValAlaThrValArg                              114511501155                                                                  GCCATGGGCCCGTACATGGAGCGGCTGGCGGGTAAACATCCAAGTATA3977                          AlaMetGlyProTyrMetGluArgLeuAlaGlyLysHisProSerIle                              116011651170                                                                  TACTGTGGGCATGATACAACTGCTTTCACAAGGATCACTGACTCCCCC4025                          TyrCysGlyHisAspThrThrAlaPheThrArgIleThrAspSerPro                              117511801185                                                                  CTGACGTATTCAACCTATGGGAGGTTTTTGGCCAACCCTAGGCAGATG4073                          LeuThrTyrSerThrTyrGlyArgPheLeuAlaAsnProArgGlnMet                              1190119512001205                                                              CTACGGGGCGTTTCGGTGGTCATTTGTGATGAGTGCCACAGTCATGAC4121                          LeuArgGlyValSerValValIleCysAspGluCysHisSerHisAsp                              121012151220                                                                  TCAACCGTGCTGTTAGGCATTGGGAGAGTCCGGGAGCTGGCGCGTGGG4169                          SerThrValLeuLeuGlyIleGlyArgValArgGluLeuAlaArgGly                              122512301235                                                                  TGCGGGGTGCAACTAGTGCTCTACGCCACCGCTACACCTCCCGGATCC4217                          CysGlyValGlnLeuValLeuTyrAlaThrAlaThrProProGlySer                              124012451250                                                                  CCTATGACGCAGCACCCTTCCATAATTGAGACAAAATTGGACGTGGGC4265                          ProMetThrGlnHisProSerIleIleGluThrLysLeuAspValGly                              125512601265                                                                  GAGATTCCCTTTTATGGGCATGGAATACCCCTCGAGCGGATGCGAACC4313                          GluIleProPheTyrGlyHisGlyIleProLeuGluArgMetArgThr                              1270127512801285                                                              GGAAGGCACCTCGTGTTCTGCCATTCTAAGGCTGAGTGCGAGCGCCTT4361                          GlyArgHisLeuValPheCysHisSerLysAlaGluCysGluArgLeu                              129012951300                                                                  GCTGGCCAGTTCTCCGCTAGGGGGGTCAATGCCATTGCCTATTATAGG4409                          AlaGlyGlnPheSerAlaArgGlyValAsnAlaIleAlaTyrTyrArg                              130513101315                                                                  GGTAAAGACAGTTCTATCATCAAGGATGGGGACCTGGTGGTCTGTGCT4457                          GlyLysAspSerSerIleIleLysAspGlyAspLeuValValCysAla                              132013251330                                                                  ACAGACGCGCTTTCCACTGGGTACACTGGAAATTTCGACTCCGTCACC4505                          ThrAspAlaLeuSerThrGlyTyrThrGlyAsnPheAspSerValThr                              133513401345                                                                  GACTGTGGATTAGTGGTGGAGGAGGTCGTTGAGGTGACCCTTGATCCC4553                          AspCysGlyLeuValValGluGluValValGluValThrLeuAspPro                              1350135513601365                                                              ACCATTACCATCTCCCTGCGGACAGTGCCTGCGTCGGCTGAACTGTCG4601                          ThrIleThrIleSerLeuArgThrValProAlaSerAlaGluLeuSer                              137013751380                                                                  ATGCAAAGACGAGGACGCACGGGTAGGGGCAGGTCTGGACGCTACTAC4649                          MetGlnArgArgGlyArgThrGlyArgGlyArgSerGlyArgTyrTyr                              138513901395                                                                  TACGCGGGGGTGGGCAAAGCCCCTGCGGGTGTGGTGCGCTCAGGTCCT4697                          TyrAlaGlyValGlyLysAlaProAlaGlyValValArgSerGlyPro                              140014051410                                                                  GTCTGGTCGGCGGTGGAAGCTGGAGTGACCTGGTACGGAATGGAACCT4745                          ValTrpSerAlaValGluAlaGlyValThrTrpTyrGlyMetGluPro                              141514201425                                                                  GACTTGACAGCTAACCTACTGAGACTTTACGACGACTGCCCTTACACC4793                          AspLeuThrAlaAsnLeuLeuArgLeuTyrAspAspCysProTyrThr                              1430143514401445                                                              GCAGCCGTCGCGGCTGATATCGGAGAAGCCGCGGTGTTCTTCTCTGGG4841                          AlaAlaValAlaAlaAspIleGlyGluAlaAlaValPhePheSerGly                              145014551460                                                                  CTCGCCCCATTGAGGATGCACCCTGATGTCAGCTGGGCAAAAGTTCGC4889                          LeuAlaProLeuArgMetHisProAspValSerTrpAlaLysValArg                              146514701475                                                                  GGCGTCAACTGGCCCCTCTTGGTGGGTGTTCAGCGGACCATGTGTCGG4937                          GlyValAsnTrpProLeuLeuValGlyValGlnArgThrMetCysArg                              148014851490                                                                  GAAACACTGTCTCCCGGCCCATCGGATGACCCCCAATGGGCAGGTCTG4985                          GluThrLeuSerProGlyProSerAspAspProGlnTrpAlaGlyLeu                              149515001505                                                                  AAGGGCCCAAATCCTGTCCCACTCCTGCTGAGGTGGGGCAATGATTTA5033                          LysGlyProAsnProValProLeuLeuLeuArgTrpGlyAsnAspLeu                              1510151515201525                                                              CCATCTAAAGTGGCCGGCCACCACATAGTGGACGACCTGGTCCGGAGA5081                          ProSerLysValAlaGlyHisHisIleValAspAspLeuValArgArg                              153015351540                                                                  CTCGGTGTGGCGGAGGGTTACGTCCGCTGCGACGCTGGGCCGATCTTG5129                          LeuGlyValAlaGluGlyTyrValArgCysAspAlaGlyProIleLeu                              154515501555                                                                  ATGATCGGTCTAGCTATCGCGGGGGGAATGATCTACGCGTCATACACC5177                          MetIleGlyLeuAlaIleAlaGlyGlyMetIleTyrAlaSerTyrThr                              156015651570                                                                  GGGTCGCTAGTGGTGGTGACAGACTGGGATGTGAAGGGGGGTGGCGCC5225                          GlySerLeuValValValThrAspTrpAspValLysGlyGlyGlyAla                              157515801585                                                                  CCCCTTTATCGGCATGGAGACCAGGCCACGCCTCAGCCGGTGGTGCAG5273                          ProLeuTyrArgHisGlyAspGlnAlaThrProGlnProValValGln                              1590159516001605                                                              GTTCCTCCGGTAGACCATCGGCCGGGGGGTGAATCAGCACCATCGGAT5321                          ValProProValAspHisArgProGlyGlyGluSerAlaProSerAsp                              161016151620                                                                  GCCAAGACAGTGACAGATGCGGTGGCAGCCATCCAGGTGGACTGCGAT5369                          AlaLysThrValThrAspAlaValAlaAlaIleGlnValAspCysAsp                              162516301635                                                                  TGGACTATCATGACTCTGTCGATCGGAGAAGTGTTGTCCTTGGCTCAG5417                          TrpThrIleMetThrLeuSerIleGlyGluValLeuSerLeuAlaGln                              164016451650                                                                  GCTAAGACGGCCGAGGCCTACACAGCAACCGCCAAGTGGCTCGCTGGC5465                          AlaLysThrAlaGluAlaTyrThrAlaThrAlaLysTrpLeuAlaGly                              165516601665                                                                  TGCTATACGGGGACGCGGGCCGTTCCCACTGTATCCATTGTTGACAAG5513                          CysTyrThrGlyThrArgAlaValProThrValSerIleValAspLys                              1670167516801685                                                              CTCTTCGCCGGAGGGTGGGCGGCTGTGGTGGGCCATTGCCACAGCGTG5561                          LeuPheAlaGlyGlyTrpAlaAlaValValGlyHisCysHisSerVal                              169016951700                                                                  ATTGCTGCGGCGGTGGCGGCCTACGGGGCTTCAAGGAGCCCGCCGTTG5609                          IleAlaAlaAlaValAlaAlaTyrGlyAlaSerArgSerProProLeu                              170517101715                                                                  GCAGCCGCGGCTTCCTACCTGATGGGGTTGGGCGTTGGAGGCAACGCT5657                          AlaAlaAlaAlaSerTyrLeuMetGlyLeuGlyValGlyGlyAsnAla                              172017251730                                                                  CAGACGCGCCTGGCGTCTGCCCTCCTATTGGGGGCTGCTGGAACCGCC5705                          GlnThrArgLeuAlaSerAlaLeuLeuLeuGlyAlaAlaGlyThrAla                              173517401745                                                                  TTGGGCACTCCTGTCGTGGGCTTGACCATGGCAGGTGCGTTCATGGGG5753                          LeuGlyThrProValValGlyLeuThrMetAlaGlyAlaPheMetGly                              1750175517601765                                                              GGGGCCAGTGTCTCCCCCTCCTTGGTCACCATTTTATTGGGGGCCGTC5801                          GlyAlaSerValSerProSerLeuValThrIleLeuLeuGlyAlaVal                              177017751780                                                                  GGAGGTTGGGAGGGTGTTGTCAACGCGGCGAGCCTAGTCTTTGACTTC5849                          GlyGlyTrpGluGlyValValAsnAlaAlaSerLeuValPheAspPhe                              178517901795                                                                  ATGGCGGGGAAACTTTCATCAGAAGATCTGTGGTATGCCATCCCGGTA5897                          MetAlaGlyLysLeuSerSerGluAspLeuTrpTyrAlaIleProVal                              180018051810                                                                  CTGACCAGCCCGGGGGCGGGCCTTGCGGGGATCGCTCTCGGGTTGGTT5945                          LeuThrSerProGlyAlaGlyLeuAlaGlyIleAlaLeuGlyLeuVal                              181518201825                                                                  TTGTATTCAGCTAACAACTCTGGCACTACCACTTGGTTGAACCGTCTG5993                          LeuTyrSerAlaAsnAsnSerGlyThrThrThrTrpLeuAsnArgLeu                              1830183518401845                                                              CTGACTACGTTACCAAGGTCTTCATGTATCCCGGACAGTTACTTTCAG6041                          LeuThrThrLeuProArgSerSerCysIleProAspSerTyrPheGln                              185018551860                                                                  CAAGTTGACTATTGCGACAAGGTCTCAGCCGTGCTCCGGCGCCTGAGC6089                          GlnValAspTyrCysAspLysValSerAlaValLeuArgArgLeuSer                              186518701875                                                                  CTCACCCGCACAGTGGTTGCCCTGGTCAACAGGGAGCCTAAGGTGGAT6137                          LeuThrArgThrValValAlaLeuValAsnArgGluProLysValAsp                              188018851890                                                                  GAGGTACAGGTGGGGTATGTCTGGGACCTGTGGGAGTGGATCATGCGC6185                          GluValGlnValGlyTyrValTrpAspLeuTrpGluTrpIleMetArg                              189519001905                                                                  CAAGTGCGCGTGGTCATGGCCAGACTCAGGGCCCTCTGCCCCGTGGTG6233                          GlnValArgValValMetAlaArgLeuArgAlaLeuCysProValVal                              1910191519201925                                                              TCACTACCCTTGTGGCATTGCGGGGAGGGGTGGTCCGGGGAATGGTTG6281                          SerLeuProLeuTrpHisCysGlyGluGlyTrpSerGlyGluTrpLeu                              193019351940                                                                  CTTGACGGTCATGTTGAGAGTCGCTGCCTCTGTGGCTGCGTGATCACT6329                          LeuAspGlyHisValGluSerArgCysLeuCysGlyCysValIleThr                              194519501955                                                                  GGTGACGTTCTGAATGGGCAACTCAAAGAACCAGTTTACTCTACCAAG6377                          GlyAspValLeuAsnGlyGlnLeuLysGluProValTyrSerThrLys                              196019651970                                                                  CTGTGCCGGCACTATTGGATGGGGACTGTCCCTGTGAACATGCTGGGT6425                          LeuCysArgHisTyrTrpMetGlyThrValProValAsnMetLeuGly                              197519801985                                                                  TACGGTGAAACGTCGCCTCTCCTGGCCTCCGACACCCCGAAGGTTGTG6473                          TyrGlyGluThrSerProLeuLeuAlaSerAspThrProLysValVal                              1990199520002005                                                              CCCTTCGGGACGTCTGGCTGGGCTGAGGTGGTGGTGACCACTACCCAC6521                          ProPheGlyThrSerGlyTrpAlaGluValValValThrThrThrHis                              201020152020                                                                  GTGGTAATCAGGAGGACCTCCGCCTATAAGCTGCTGCGCCAGCAAATC6569                          ValValIleArgArgThrSerAlaTyrLysLeuLeuArgGlnGlnIle                              202520302035                                                                  CTATCGGCTGCTGTAGCTGAGCCCTACTACGTCGACGGCATTCCGGTC6617                          LeuSerAlaAlaValAlaGluProTyrTyrValAspGlyIleProVal                              204020452050                                                                  TCATGGGACGCGGACGCTCGTGCGCCCGCCATGGTCTATGGCCCTGGG6665                          SerTrpAspAlaAspAlaArgAlaProAlaMetValTyrGlyProGly                              205520602065                                                                  CAAAGTGTTACCATTGACGGGGAGCGCTACACCTTGCCTCATCAACTG6713                          GlnSerValThrIleAspGlyGluArgTyrThrLeuProHisGlnLeu                              2070207520802085                                                              AGGCTCAGGAATGTGGCACCCTCTGAGGTTTCATCCGAGGTGTCCATT6761                          ArgLeuArgAsnValAlaProSerGluValSerSerGluValSerIle                              209020952100                                                                  GACATTGGGACGGAGACTGAAGACTCAGAACTGACTGAGGCCGATCTG6809                          AspIleGlyThrGluThrGluAspSerGluLeuThrGluAlaAspLeu                              210521102115                                                                  CCGCCGGCGGCTGCTGCTCTCCAAGCGATCGAGAATGCTGCGAGGATT6857                          ProProAlaAlaAlaAlaLeuGlnAlaIleGluAsnAlaAlaArgIle                              212021252130                                                                  CTTGAACCGCACATTGATGTCATCATGGAGGACTGCAGTACACCCTCT6905                          LeuGluProHisIleAspValIleMetGluAspCysSerThrProSer                              213521402145                                                                  CTTTGTGGTAGTAGCCGAGAGATGCCTGTATGGGGAGAAGACATCCCC6953                          LeuCysGlySerSerArgGluMetProValTrpGlyGluAspIlePro                              2150215521602165                                                              CGTACTCCATCGCCAGCACTTATCTCGGTTACTGAGAGCAGCTCAGAT7001                          ArgThrProSerProAlaLeuIleSerValThrGluSerSerSerAsp                              217021752180                                                                  GAGAAGACCCCGTCGGTGTCCTCCTCGCAGGAGGATACCCCGTCCTCT7049                          GluLysThrProSerValSerSerSerGlnGluAspThrProSerSer                              218521902195                                                                  GACTCATTCGAGGTCATCCAAGAGTCCGAGACAGCCGAAGGGGAGGAA7097                          AspSerPheGluValIleGlnGluSerGluThrAlaGluGlyGluGlu                              220022052210                                                                  AGTGTCTTCAACGTGGCTCTTTCCGTATTAAAAGCCTTATTTCCACAG7145                          SerValPheAsnValAlaLeuSerValLeuLysAlaLeuPheProGln                              221522202225                                                                  AGCGACGCGACCAGGAAGCTTACCGTCAAGATGTCGTGCTGCGTTGAA7193                          SerAspAlaThrArgLysLeuThrValLysMetSerCysCysValGlu                              2230223522402245                                                              AAGAGCGTCACGCGCTTTTTCTCATTGGGGTTGACGGTGGCTGATGTT7241                          LysSerValThrArgPhePheSerLeuGlyLeuThrValAlaAspVal                              225022552260                                                                  GCTAGCCTGTGTGAGATGGAAATCCAGAACCATACAGCCTATTGTGAC7289                          AlaSerLeuCysGluMetGluIleGlnAsnHisThrAlaTyrCysAsp                              226522702275                                                                  CAGGTGCGCACTCCGCTTGAATTGCAGGTTGGGTGCTTGGTGGGCAAT7337                          GlnValArgThrProLeuGluLeuGlnValGlyCysLeuValGlyAsn                              228022852290                                                                  GAACTTACCTTTGAATGTGACAAGTGTGAGGCTAGGCAAGAAACCTTG7385                          GluLeuThrPheGluCysAspLysCysGluAlaArgGlnGluThrLeu                              229523002305                                                                  GCCTCCTTCTCTTACATTTGGTCTGGAGTGCCGCTGACTAGGGCCACG7433                          AlaSerPheSerTyrIleTrpSerGlyValProLeuThrArgAlaThr                              2310231523202325                                                              CCGGCCAAGCCTCCCGTGGTGAGGCCGGTTGGCTCTTTGTTAGTGGCC7481                          ProAlaLysProProValValArgProValGlySerLeuLeuValAla                              233023352340                                                                  GACACTACTAAGGTGTATGTTACCAATCCAGACAATGTGGGACGGAGG7529                          AspThrThrLysValTyrValThrAsnProAspAsnValGlyArgArg                              234523502355                                                                  GTGGACAAGGTGACCTTCTGGCGTGCTCCTAGGGTTCATGATAAGTAC7577                          ValAspLysValThrPheTrpArgAlaProArgValHisAspLysTyr                              236023652370                                                                  CTCGTGGACTCTATTGAGCGCGCTAAGAGGGCCGCTCAAGCCTGCCTA7625                          LeuValAspSerIleGluArgAlaLysArgAlaAlaGlnAlaCysLeu                              237523802385                                                                  AGCATGGGTTACACTTATGAGGAAGCAATAAGGACTGTAAGGCCACAT7673                          SerMetGlyTyrThrTyrGluGluAlaIleArgThrValArgProHis                              2390239524002405                                                              GCTGCCATGGGCTGGGGATCTAAGGTGTCGGTTAAGGACTTAGCCACC7721                          AlaAlaMetGlyTrpGlySerLysValSerValLysAspLeuAlaThr                              241024152420                                                                  CCCGCGGGGAAGATGGCCGTCCATGACCGGCTTCAGGAGATACTTGAA7769                          ProAlaGlyLysMetAlaValHisAspArgLeuGlnGluIleLeuGlu                              242524302435                                                                  GGGACTCCGGTCCCCTTTACTCTTACTGTGAAAAAGGAGGTGTTCTTC7817                          GlyThrProValProPheThrLeuThrValLysLysGluValPhePhe                              244024452450                                                                  AAAGACCGGAAGGAGGAGAAGGCCCCCCGCCTCATTGTGTTCCCCCCC7865                          LysAspArgLysGluGluLysAlaProArgLeuIleValPheProPro                              245524602465                                                                  CTGGACTTCCGGATAGCTGAAAAGCTCATCTTGGGAGACCCAGGCCGG7913                          LeuAspPheArgIleAlaGluLysLeuIleLeuGlyAspProGlyArg                              2470247524802485                                                              GTAGCCAAGGCGGTGTTGGGGGGGGCCTACGCCTTCCAGTACACCCCA7961                          ValAlaLysAlaValLeuGlyGlyAlaTyrAlaPheGlnTyrThrPro                              249024952500                                                                  AATCAGCGAGTTAAGGAGATGCTCAAGCTATGGGAGTCTAAGAAGACC8009                          AsnGlnArgValLysGluMetLeuLysLeuTrpGluSerLysLysThr                              250525102515                                                                  CCTTGCGCCATCTGTGTGGACGCCACCTGCTTCGACAGTAGCATAACT8057                          ProCysAlaIleCysValAspAlaThrCysPheAspSerSerIleThr                              252025252530                                                                  GAAGAGGACGTGGCTTTGGAGACAGAGCTATACGCTCTGGCCTCTGAC8105                          GluGluAspValAlaLeuGluThrGluLeuTyrAlaLeuAlaSerAsp                              253525402545                                                                  CATCCAGAATGGGTGCGGGCACTTGGGAAATACTATGCCTCAGGCACC8153                          HisProGluTrpValArgAlaLeuGlyLysTyrTyrAlaSerGlyThr                              2550255525602565                                                              ATGGTCACCCCGGAAGGGGTGCCCGTCGGTGAGAGGTATTGCAGATCC8201                          MetValThrProGluGlyValProValGlyGluArgTyrCysArgSer                              257025752580                                                                  TCGGGTGTCCTAACAACTAGCGCGAGCAACTGCTTGACCTGCTACATC8249                          SerGlyValLeuThrThrSerAlaSerAsnCysLeuThrCysTyrIle                              258525902595                                                                  AAGGTGAAAGCTGCCTGTGAGAGAGTGGGGCTGAAAAATGTCTCTCTT8297                          LysValLysAlaAlaCysGluArgValGlyLeuLysAsnValSerLeu                              260026052610                                                                  CTCATAGCCGGCGATGACTGCTTGATCATATGTGAGCGGCCAGTGTGC8345                          LeuIleAlaGlyAspAspCysLeuIleIleCysGluArgProValCys                              261526202625                                                                  GACCCAAGCGACGCTTTGGGCAGAGCCCTAGCGAGCTATGGGTACGCG8393                          AspProSerAspAlaLeuGlyArgAlaLeuAlaSerTyrGlyTyrAla                              2630263526402645                                                              TGCGAGCCCTCATATCATGCATCATTGGACACGGCCCCCTTCTGCTCC8441                          CysGluProSerTyrHisAlaSerLeuAspThrAlaProPheCysSer                              265026552660                                                                  ACTTGGCTTGCTGAGTGCAATGCAGATGGGAAGCGCCATTTCTTCCTG8489                          ThrTrpLeuAlaGluCysAsnAlaAspGlyLysArgHisPhePheLeu                              266526702675                                                                  ACCACGGACTTCCGGAGGCCGCTCGCTCGCATGTCGAGTGAGTATAGT8537                          ThrThrAspPheArgArgProLeuAlaArgMetSerSerGluTyrSer                              268026852690                                                                  GACCCGATGGCTTCGGCGATCGGTTACATCCTCCTTTATCCTTGGCAC8585                          AspProMetAlaSerAlaIleGlyTyrIleLeuLeuTyrProTrpHis                              269527002705                                                                  CCCATCACACGGTGGGTCATCATCCCTCATGTGCTAACGTGCGCATTC8633                          ProIleThrArgTrpValIleIleProHisValLeuThrCysAlaPhe                              2710271527202725                                                              AGGGGTGGAGGCACACCGTCTGATCCGGTTTGGTGCCAGGTGCATGGT8681                          ArgGlyGlyGlyThrProSerAspProValTrpCysGlnValHisGly                              273027352740                                                                  AACTACTACAAGTTTCCACTGGACAAACTGCCTAACATCATCGTGGCC8729                          AsnTyrTyrLysPheProLeuAspLysLeuProAsnIleIleValAla                              274527502755                                                                  CTCCACGGACCAGCAGCGTTGAGGGTTACCGCAGACACAACTAAAACA8777                          LeuHisGlyProAlaAlaLeuArgValThrAlaAspThrThrLysThr                              276027652770                                                                  AAGATGGAGGCTGGTAAGGTTCTGAGCGACCTCAAGCTCCCTGGCTTA8825                          LysMetGluAlaGlyLysValLeuSerAspLeuLysLeuProGlyLeu                              277527802785                                                                  GCAGTCCACCGAAAGAAGGCCGGGGCGTTGCGAACACGCATGCTCCGC8873                          AlaValHisArgLysLysAlaGlyAlaLeuArgThrArgMetLeuArg                              2790279528002805                                                              TCGCGCGGTTGGGCTGAGTTGGCTAGGGGCTTGTTGTGGCATCCAGGC8921                          SerArgGlyTrpAlaGluLeuAlaArgGlyLeuLeuTrpHisProGly                              281028152820                                                                  CTACGGCTTCCTCCCCCTGAGATTGCTGGTATCCCGGGGGGTTTCCCT8969                          LeuArgLeuProProProGluIleAlaGlyIleProGlyGlyPhePro                              282528302835                                                                  CTCTCCCCCCCCTATATGGGGGTGGTACATCAATTGGATTTCACAAGC9017                          LeuSerProProTyrMetGlyValValHisGlnLeuAspPheThrSer                              284028452850                                                                  CAGAGGAGTCGCTGGCGGTGGTTGGGGTTCTTAGCCCTGCTCATCGTA9065                          GlnArgSerArgTrpArgTrpLeuGlyPheLeuAlaLeuLeuIleVal                              285528602865                                                                  GCCCTCTTCGGGTGAACTAAATTCATCTGTTGCGGCAAGGTCTGGTGACTGA9117                      AlaLeuPheGly                                                                  2870                                                                          TCATCACCGGAGGAGGTTCCCGCCCTCCCCGCCCCAGGGGTCTCCCCGCTGGGTAAAAAG9177              GGCCCGGCCTTGGGAGGCATGGTGGTTACTAACCCCCTGGCAGGGTCAAAGCCTGATGGT9237              GCTAATGCACTGCCACTTCGGTGGCGGGTCGCTACCTTATAGCGTAATCCGTGACTACGG9297              GCTGCTCGCAGAGCCCTCCCCGGATGGGGCACAGTGCACTGTGATCTGAAGGGGTGCACC9357              CCGGGAAGAGCTCGGCCCGAAGGCCGGSTTCTACT9392                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2873 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      MetGlyProProSerSerAlaAlaAlaCysSerArgGlySerProArg                              151015                                                                        IleLeuArgValArgAlaGlyGlyIleSerPhePheTyrThrIleMet                              202530                                                                        AlaValLeuLeuLeuLeuLeuValValGluAlaGlyAlaIleLeuAla                              354045                                                                        ProAlaThrHisAlaCysArgAlaAsnGlyGlnTyrPheLeuThrAsn                              505560                                                                        CysCysAlaProGluAspIleGlyPheCysLeuGluGlyGlyCysLeu                              65707580                                                                      ValAlaLeuGlyCysThrIleCysThrAspGlnCysTrpProLeuTyr                              859095                                                                        GlnAlaGlyLeuAlaValArgProGlyLysSerAlaAlaGlnLeuVal                              100105110                                                                     GlyGluLeuGlySerLeuTyrGlyProLeuSerValSerAlaTyrVal                              115120125                                                                     AlaGlyIleLeuGlyLeuGlyGluValTyrSerGlyValLeuThrVal                              130135140                                                                     GlyValAlaLeuThrArgArgValTyrProValProAsnLeuThrCys                              145150155160                                                                  AlaValAlaCysGluLeuLysTrpGluSerGluPheTrpArgTrpThr                              165170175                                                                     GluGlnLeuAlaSerAsnTyrTrpIleLeuGluTyrLeuTrpLysVal                              180185190                                                                     ProPheAspPheTrpArgGlyValIleSerLeuThrProLeuLeuVal                              195200205                                                                     CysValAlaAlaLeuLeuLeuLeuGluGlnArgIleValMetValPhe                              210215220                                                                     LeuLeuValThrMetAlaGlyMetSerGlnGlyAlaProAlaSerVal                              225230235240                                                                  LeuGlySerArgProPheAspTyrGlyLeuThrTrpGlnThrCysSer                              245250255                                                                     CysArgAlaAsnGlySerArgPheSerThrGlyGluLysValTrpAsp                              260265270                                                                     ArgGlyAsnValThrLeuGlnCysAspCysProAsnGlyProTrpVal                              275280285                                                                     TrpLeuProAlaPheCysGlnAlaIleGlyTrpGlyAspProIleThr                              290295300                                                                     TyrTrpSerHisGlyGlnAsnGlnTrpProLeuSerCysProGlnTyr                              305310315320                                                                  ValTyrGlySerAlaThrValThrCysValTrpGlySerAlaSerTrp                              325330335                                                                     PheAlaSerThrSerGlyArgAspSerLysIleAspValTrpSerLeu                              340345350                                                                     ValProValGlySerAlaThrCysThrIleAlaAlaLeuGlySerSer                              355360365                                                                     AspArgAspThrValProGlyLeuSerGluTrpGlyIleProCysVal                              370375380                                                                     ThrCysValLeuAspArgArgProAlaSerCysGlyThrCysValArg                              385390395400                                                                  AspCysTrpProGluThrGlySerValArgPheProPheHisArgCys                              405410415                                                                     GlyValGlyProArgLeuThrLysAspLeuGluAlaValProPheVal                              420425430                                                                     AsnArgThrThrProPheThrIleArgGlyProLeuGlyAsnGlnGly                              435440445                                                                     ArgGlyAsnProValArgSerProLeuGlyPheGlySerTyrAlaMet                              450455460                                                                     ThrArgIleArgAspThrLeuHisLeuValGluCysProThrProAla                              465470475480                                                                  IleGluProProThrGlyThrPheGlyPhePheProGlyThrProPro                              485490495                                                                     LeuAsnAsnCysMetLeuLeuGlyThrGluValSerGluAlaLeuGly                              500505510                                                                     GlyAlaGlyLeuThrGlyGlyPheTyrGluProLeuValArgArgCys                              515520525                                                                     SerLysLeuMetGlySerArgAsnProValCysProGlyPheAlaTrp                              530535540                                                                     LeuSerSerGlyArgProAspGlyPheIleHisValGlnGlyHisLeu                              545550555560                                                                  GlnGluValAspAlaGlyAsnPheIleProProProArgTrpLeuLeu                              565570575                                                                     LeuAspPheValPheValLeuLeuTyrLeuMetLysLeuAlaGluAla                              580585590                                                                     ArgLeuValProLeuIleLeuLeuLeuLeuTrpTrpTrpValAsnGln                              595600605                                                                     LeuAlaValLeuGlyLeuProAlaValGluAlaAlaValAlaGlyGlu                              610615620                                                                     ValPheAlaGlyProAlaLeuSerTrpCysLeuGlyLeuProValVal                              625630635640                                                                  SerMetIleLeuGlyLeuAlaAsnLeuValLeuTyrPheArgTrpLeu                              645650655                                                                     GlyProGlnArgLeuMetPheLeuValLeuTrpLysLeuAlaArgGly                              660665670                                                                     AlaPheProLeuAlaLeuLeuMetGlyIleSerAlaThrArgGlyArg                              675680685                                                                     ThrSerValLeuGlyAlaGluPheCysPheAspAlaThrPheGluVal                              690695700                                                                     AspThrSerValLeuGlyTrpValValAlaSerValValAlaTrpAla                              705710715720                                                                  IleAlaLeuLeuSerSerMetSerAlaGlyGlyTrpArgHisLysAla                              725730735                                                                     ValIleTyrArgThrTrpCysLysGlyTyrGlnAlaIleArgGlnArg                              740745750                                                                     ValValArgSerProLeuGlyGluGlyArgProAlaLysProLeuThr                              755760765                                                                     PheAlaTrpCysLeuAlaSerTyrIleTrpProAspAlaValMetMet                              770775780                                                                     ValValValAlaLeuValLeuLeuPheGlyLeuPheAspAlaLeuAsp                              785790795800                                                                  TrpAlaLeuGluGluIleLeuValSerArgProSerLeuArgArgLeu                              805810815                                                                     AlaArgValValGluCysCysValMetAlaGlyGluLysAlaThrThr                              820825830                                                                     ValArgLeuValSerLysMetCysAlaArgGlyAlaTyrLeuPheAsp                              835840845                                                                     HisMetGlySerPheSerArgAlaValLysGluArgLeuLeuGluTrp                              850855860                                                                     AspAlaAlaLeuGluProLeuSerPheThrArgThrAspCysArgIle                              865870875880                                                                  IleArgAspAlaAlaArgThrLeuSerCysGlyGlnCysValMetGly                              885890895                                                                     LeuProValValAlaArgArgGlyAspGluValLeuIleGlyValPhe                              900905910                                                                     GlnAspValAsnHisLeuProProGlyPheValProThrAlaProVal                              915920925                                                                     ValIleArgArgCysGlyLysGlyPheLeuGlyValThrLysAlaAla                              930935940                                                                     LeuThrGlyArgAspProAspLeuHisProGlyAsnValMetValLeu                              945950955960                                                                  GlyThrAlaThrSerArgSerMetGlyThrCysLeuAsnGlyLeuLeu                              965970975                                                                     PheThrThrPheHisGlyAlaSerSerArgThrIleAlaThrProVal                              980985990                                                                     GlyAlaLeuAsnProArgTrpTrpSerAlaSerAspAspValThrVal                              99510001005                                                                   TyrProLeuProAspGlyAlaThrSerLeuThrProCysThrCysGln                              101010151020                                                                  AlaGluSerCysTrpValIleArgSerAspGlyAlaLeuCysHisGly                              1025103010351040                                                              LeuSerLysGlyAspLysValGluLeuAspValAlaMetGluValSer                              104510501055                                                                  AspPheArgGlySerSerGlySerProValLeuCysAspGluGlyHis                              106010651070                                                                  AlaValGlyMetLeuValSerValLeuHisSerGlyGlyArgValThr                              107510801085                                                                  AlaAlaArgPheThrArgProTrpThrGlnValProThrAspAlaLys                              109010951100                                                                  ThrThrThrGluProProProValProAlaLysGlyValPheLysGlu                              1105111011151120                                                              AlaProLeuPheMetProThrGlyAlaGlyLysSerThrArgValPro                              112511301135                                                                  LeuGluTyrAspAsnMetGlyHisLysValLeuIleLeuAsnProSer                              114011451150                                                                  ValAlaThrValArgAlaMetGlyProTyrMetGluArgLeuAlaGly                              115511601165                                                                  LysHisProSerIleTyrCysGlyHisAspThrThrAlaPheThrArg                              117011751180                                                                  IleThrAspSerProLeuThrTyrSerThrTyrGlyArgPheLeuAla                              1185119011951200                                                              AsnProArgGlnMetLeuArgGlyValSerValValIleCysAspGlu                              120512101215                                                                  CysHisSerHisAspSerThrValLeuLeuGlyIleGlyArgValArg                              122012251230                                                                  GluLeuAlaArgGlyCysGlyValGlnLeuValLeuTyrAlaThrAla                              123512401245                                                                  ThrProProGlySerProMetThrGlnHisProSerIleIleGluThr                              125012551260                                                                  LysLeuAspValGlyGluIleProPheTyrGlyHisGlyIleProLeu                              1265127012751280                                                              GluArgMetArgThrGlyArgHisLeuValPheCysHisSerLysAla                              128512901295                                                                  GluCysGluArgLeuAlaGlyGlnPheSerAlaArgGlyValAsnAla                              130013051310                                                                  IleAlaTyrTyrArgGlyLysAspSerSerIleIleLysAspGlyAsp                              131513201325                                                                  LeuValValCysAlaThrAspAlaLeuSerThrGlyTyrThrGlyAsn                              133013351340                                                                  PheAspSerValThrAspCysGlyLeuValValGluGluValValGlu                              1345135013551360                                                              ValThrLeuAspProThrIleThrIleSerLeuArgThrValProAla                              136513701375                                                                  SerAlaGluLeuSerMetGlnArgArgGlyArgThrGlyArgGlyArg                              138013851390                                                                  SerGlyArgTyrTyrTyrAlaGlyValGlyLysAlaProAlaGlyVal                              139514001405                                                                  ValArgSerGlyProValTrpSerAlaValGluAlaGlyValThrTrp                              141014151420                                                                  TyrGlyMetGluProAspLeuThrAlaAsnLeuLeuArgLeuTyrAsp                              1425143014351440                                                              AspCysProTyrThrAlaAlaValAlaAlaAspIleGlyGluAlaAla                              144514501455                                                                  ValPhePheSerGlyLeuAlaProLeuArgMetHisProAspValSer                              146014651470                                                                  TrpAlaLysValArgGlyValAsnTrpProLeuLeuValGlyValGln                              147514801485                                                                  ArgThrMetCysArgGluThrLeuSerProGlyProSerAspAspPro                              149014951500                                                                  GlnTrpAlaGlyLeuLysGlyProAsnProValProLeuLeuLeuArg                              1505151015151520                                                              TrpGlyAsnAspLeuProSerLysValAlaGlyHisHisIleValAsp                              152515301535                                                                  AspLeuValArgArgLeuGlyValAlaGluGlyTyrValArgCysAsp                              154015451550                                                                  AlaGlyProIleLeuMetIleGlyLeuAlaIleAlaGlyGlyMetIle                              155515601565                                                                  TyrAlaSerTyrThrGlySerLeuValValValThrAspTrpAspVal                              157015751580                                                                  LysGlyGlyGlyAlaProLeuTyrArgHisGlyAspGlnAlaThrPro                              1585159015951600                                                              GlnProValValGlnValProProValAspHisArgProGlyGlyGlu                              160516101615                                                                  SerAlaProSerAspAlaLysThrValThrAspAlaValAlaAlaIle                              162016251630                                                                  GlnValAspCysAspTrpThrIleMetThrLeuSerIleGlyGluVal                              163516401645                                                                  LeuSerLeuAlaGlnAlaLysThrAlaGluAlaTyrThrAlaThrAla                              165016551660                                                                  LysTrpLeuAlaGlyCysTyrThrGlyThrArgAlaValProThrVal                              1665167016751680                                                              SerIleValAspLysLeuPheAlaGlyGlyTrpAlaAlaValValGly                              168516901695                                                                  HisCysHisSerValIleAlaAlaAlaValAlaAlaTyrGlyAlaSer                              170017051710                                                                  ArgSerProProLeuAlaAlaAlaAlaSerTyrLeuMetGlyLeuGly                              171517201725                                                                  ValGlyGlyAsnAlaGlnThrArgLeuAlaSerAlaLeuLeuLeuGly                              173017351740                                                                  AlaAlaGlyThrAlaLeuGlyThrProValValGlyLeuThrMetAla                              1745175017551760                                                              GlyAlaPheMetGlyGlyAlaSerValSerProSerLeuValThrIle                              176517701775                                                                  LeuLeuGlyAlaValGlyGlyTrpGluGlyValValAsnAlaAlaSer                              178017851790                                                                  LeuValPheAspPheMetAlaGlyLysLeuSerSerGluAspLeuTrp                              179518001805                                                                  TyrAlaIleProValLeuThrSerProGlyAlaGlyLeuAlaGlyIle                              181018151820                                                                  AlaLeuGlyLeuValLeuTyrSerAlaAsnAsnSerGlyThrThrThr                              1825183018351840                                                              TrpLeuAsnArgLeuLeuThrThrLeuProArgSerSerCysIlePro                              184518501855                                                                  AspSerTyrPheGlnGlnValAspTyrCysAspLysValSerAlaVal                              186018651870                                                                  LeuArgArgLeuSerLeuThrArgThrValValAlaLeuValAsnArg                              187518801885                                                                  GluProLysValAspGluValGlnValGlyTyrValTrpAspLeuTrp                              189018951900                                                                  GluTrpIleMetArgGlnValArgValValMetAlaArgLeuArgAla                              1905191019151920                                                              LeuCysProValValSerLeuProLeuTrpHisCysGlyGluGlyTrp                              192519301935                                                                  SerGlyGluTrpLeuLeuAspGlyHisValGluSerArgCysLeuCys                              194019451950                                                                  GlyCysValIleThrGlyAspValLeuAsnGlyGlnLeuLysGluPro                              195519601965                                                                  ValTyrSerThrLysLeuCysArgHisTyrTrpMetGlyThrValPro                              197019751980                                                                  ValAsnMetLeuGlyTyrGlyGluThrSerProLeuLeuAlaSerAsp                              1985199019952000                                                              ThrProLysValValProPheGlyThrSerGlyTrpAlaGluValVal                              200520102015                                                                  ValThrThrThrHisValValIleArgArgThrSerAlaTyrLysLeu                              202020252030                                                                  LeuArgGlnGlnIleLeuSerAlaAlaValAlaGluProTyrTyrVal                              203520402045                                                                  AspGlyIleProValSerTrpAspAlaAspAlaArgAlaProAlaMet                              205020552060                                                                  ValTyrGlyProGlyGlnSerValThrIleAspGlyGluArgTyrThr                              2065207020752080                                                              LeuProHisGlnLeuArgLeuArgAsnValAlaProSerGluValSer                              208520902095                                                                  SerGluValSerIleAspIleGlyThrGluThrGluAspSerGluLeu                              210021052110                                                                  ThrGluAlaAspLeuProProAlaAlaAlaAlaLeuGlnAlaIleGlu                              211521202125                                                                  AsnAlaAlaArgIleLeuGluProHisIleAspValIleMetGluAsp                              213021352140                                                                  CysSerThrProSerLeuCysGlySerSerArgGluMetProValTrp                              2145215021552160                                                              GlyGluAspIleProArgThrProSerProAlaLeuIleSerValThr                              216521702175                                                                  GluSerSerSerAspGluLysThrProSerValSerSerSerGlnGlu                              218021852190                                                                  AspThrProSerSerAspSerPheGluValIleGlnGluSerGluThr                              219522002205                                                                  AlaGluGlyGluGluSerValPheAsnValAlaLeuSerValLeuLys                              221022152220                                                                  AlaLeuPheProGlnSerAspAlaThrArgLysLeuThrValLysMet                              2225223022352240                                                              SerCysCysValGluLysSerValThrArgPhePheSerLeuGlyLeu                              224522502255                                                                  ThrValAlaAspValAlaSerLeuCysGluMetGluIleGlnAsnHis                              226022652270                                                                  ThrAlaTyrCysAspGlnValArgThrProLeuGluLeuGlnValGly                              227522802285                                                                  CysLeuValGlyAsnGluLeuThrPheGluCysAspLysCysGluAla                              229022952300                                                                  ArgGlnGluThrLeuAlaSerPheSerTyrIleTrpSerGlyValPro                              2305231023152320                                                              LeuThrArgAlaThrProAlaLysProProValValArgProValGly                              232523302335                                                                  SerLeuLeuValAlaAspThrThrLysValTyrValThrAsnProAsp                              234023452350                                                                  AsnValGlyArgArgValAspLysValThrPheTrpArgAlaProArg                              235523602365                                                                  ValHisAspLysTyrLeuValAspSerIleGluArgAlaLysArgAla                              237023752380                                                                  AlaGlnAlaCysLeuSerMetGlyTyrThrTyrGluGluAlaIleArg                              2385239023952400                                                              ThrValArgProHisAlaAlaMetGlyTrpGlySerLysValSerVal                              240524102415                                                                  LysAspLeuAlaThrProAlaGlyLysMetAlaValHisAspArgLeu                              242024252430                                                                  GlnGluIleLeuGluGlyThrProValProPheThrLeuThrValLys                              243524402445                                                                  LysGluValPhePheLysAspArgLysGluGluLysAlaProArgLeu                              245024552460                                                                  IleValPheProProLeuAspPheArgIleAlaGluLysLeuIleLeu                              2465247024752480                                                              GlyAspProGlyArgValAlaLysAlaValLeuGlyGlyAlaTyrAla                              248524902495                                                                  PheGlnTyrThrProAsnGlnArgValLysGluMetLeuLysLeuTrp                              250025052510                                                                  GluSerLysLysThrProCysAlaIleCysValAspAlaThrCysPhe                              251525202525                                                                  AspSerSerIleThrGluGluAspValAlaLeuGluThrGluLeuTyr                              253025352540                                                                  AlaLeuAlaSerAspHisProGluTrpValArgAlaLeuGlyLysTyr                              2545255025552560                                                              TyrAlaSerGlyThrMetValThrProGluGlyValProValGlyGlu                              256525702575                                                                  ArgTyrCysArgSerSerGlyValLeuThrThrSerAlaSerAsnCys                              258025852590                                                                  LeuThrCysTyrIleLysValLysAlaAlaCysGluArgValGlyLeu                              259526002605                                                                  LysAsnValSerLeuLeuIleAlaGlyAspAspCysLeuIleIleCys                              261026152620                                                                  GluArgProValCysAspProSerAspAlaLeuGlyArgAlaLeuAla                              2625263026352640                                                              SerTyrGlyTyrAlaCysGluProSerTyrHisAlaSerLeuAspThr                              264526502655                                                                  AlaProPheCysSerThrTrpLeuAlaGluCysAsnAlaAspGlyLys                              266026652670                                                                  ArgHisPhePheLeuThrThrAspPheArgArgProLeuAlaArgMet                              267526802685                                                                  SerSerGluTyrSerAspProMetAlaSerAlaIleGlyTyrIleLeu                              269026952700                                                                  LeuTyrProTrpHisProIleThrArgTrpValIleIleProHisVal                              2705271027152720                                                              LeuThrCysAlaPheArgGlyGlyGlyThrProSerAspProValTrp                              272527302735                                                                  CysGlnValHisGlyAsnTyrTyrLysPheProLeuAspLysLeuPro                              274027452750                                                                  AsnIleIleValAlaLeuHisGlyProAlaAlaLeuArgValThrAla                              275527602765                                                                  AspThrThrLysThrLysMetGluAlaGlyLysValLeuSerAspLeu                              277027752780                                                                  LysLeuProGlyLeuAlaValHisArgLysLysAlaGlyAlaLeuArg                              2785279027952800                                                              ThrArgMetLeuArgSerArgGlyTrpAlaGluLeuAlaArgGlyLeu                              280528102815                                                                  LeuTrpHisProGlyLeuArgLeuProProProGluIleAlaGlyIle                              282028252830                                                                  ProGlyGlyPheProLeuSerProProTyrMetGlyValValHisGln                              283528402845                                                                  LeuAspPheThrSerGlnArgSerArgTrpArgTrpLeuGlyPheLeu                              285028552860                                                                  AlaLeuLeuIleValAlaLeuPheGly                                                   28652870                                                                      (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PROBE 470-20- 1-152F                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TCGGTTACTGAGAGCAGCTCAGATGAG27                                                 (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: JML-A, PRIMER                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AGGAATTCAGCGGCCGCGAG20                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: JML-B, PRIMER                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CTCGCGGCCGCTGAATTCCTTT22                                                      (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 203 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 470-20-1 CLONE, WITHOUT SISPA                         LINKERS                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..203                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GGCTGTCTCGGACTCTTGGATGACCTCGAATGAGTCAGAGGACGGG46                              AlaValSerAspSerTrpMetThrSerAsnGluSerGluAspGly                                 151015                                                                        GTATCCTCCTGCGAGGAGGACACCGGCGGGGTCTTCTCATCTGAGCTG94                            ValSerSerCysGluGluAspThrGlyGlyValPheSerSerGluLeu                              202530                                                                        CTCTCAGTAACCGAGATAAGTGCTGGCGATGGAGTACGGGGGATGTCT142                           LeuSerValThrGluIleSerAlaGlyAspGlyValArgGlyMetSer                              354045                                                                        TCTCCCCATACAGGCATCTCTCGGCTACTACCACAAAGAGAGGGTGTA190                           SerProHisThrGlyIleSerArgLeuLeuProGlnArgGluGlyVal                              505560                                                                        CTGCAGTCCTCCA203                                                              LeuGlnSerSer                                                                  65                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AlaValSerAspSerTrpMetThrSerAsnGluSerGluAspGlyVal                              151015                                                                        SerSerCysGluGluAspThrGlyGlyValPheSerSerGluLeuLeu                              202530                                                                        SerValThrGluIleSerAlaGlyAspGlyValArgGlyMetSerSer                              354045                                                                        ProHisThrGlyIleSerArgLeuLeuProGlnArgGluGlyValLeu                              505560                                                                        GlnSerSer                                                                     65                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 470-20-1- 152R                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CTCATCTGAGCTGCTCTCAGTAACCGA27                                                 (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: OLIGONUCLEOTIDE B                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CTGTCTCGGACTCTTGGATGACCT24                                                    (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: COGNATE OLIGONUCLEOTIDE 211R'                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ATACCCCGTCCTCTGACTCATTCG24                                                    (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: COGNATE OLIGONUCLEOTIDE B'                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AGGTCATCCAAGAGTCCGAGACAG24                                                    (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: LAMBDA GT 11 FORWARD PRIMER, 20mer                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CACATGGCTGAATATCGACG20                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 4E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GCGAGCCTAGTCTTTGACTTCATGGCGGGGAAACTTTCATCAGAAGATCTGTGGTATGCC60                ATCCCGGTACTGACCAGCCCGGGGGCGGGCCTTGCGGGGATCGCTCTCGGGTTGGTTTTG120               TATTCAGCTAACAACTCTGGCACTACCACTTGGTTGAACCGTCTGCTGACTACGTTACCA180               (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 430 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 3E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GGCACTACCACTTGGTTGAACCGTCTGCTGACTACGTTACCAAGGTCTTCATGTATCCCG60                GACAGTTACTTTCAGCAAGTTGACTATTGCGACAAGGTCTCAGCCGTGCTCCGGCGCCTG120               AGCCTCACCCGCACAGTGGTTGCCCTGGTCAACAGGGAGCCTAAGGTGGATGAGGTACAG180               GTGGGGTATGTCTGGGACCTGTGGGAGTGGATCATGCGCCAAGTGCGCGTGGTCATGGCC240               AGACTCAGGGCCCTCTGCCCCGTGGTGTCACTACCCTTGTGGCATTGCGGGGAGGGGTGG300               TCCGGGGAATGGTTGCTTGACGGTCATGTTGAGAGTCGCTGCCTCTGTGGCTGCGTGATC360               ACTGGTGACGTTCTGAATGGGCAACTCAAAGAACCAGTTTACTCTACCAAGCTGTGCCGG420               CACTATTGGA430                                                                 (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 180 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 2E5                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CTTACCGTCAAGATGTCGTGCTGCGTTGAAAAGAGCGTCACGCGCTTTTTCTCATTGGGG60                TTGACGGTGGCTGATGTTGCTAGCCTGTGTGAGATGGAAATCCAGAACCATACAGCCTAT120               TGTGACCAGGTGCGCACTCCGCTTGAATTGCAGGTTGGGTGCTTGGTGGGCAATGAACTT180               (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 344 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 1E5                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      CTTCTCTTTGTGGTAGTAGCCGAGAGATGCCTGTATGGGGAGAAGACATCCCCCGTACTC60                CATCGCCAGCACTTATCTCGGTTACTGAGAGCAGCTCAGATGAGAAGACCCCGTCGGTGT120               CCTCCTCGCAGGAGGATACCCCGTCCTCTGACTCATTCGAGGTCATCCAAGAGTCCGAGA180               CAGCCGAAGGGGAGGAAAGTGTCTTCAACGTGGCTCTTTCCGTATTAAAAGCCTTATTTC240               CACAGAGCGACGCGACCAGGAAGCTTACCGTCAAGATGTCGTGCTGCGTTGAAAAGAGCG300               TCACGCGCTTTTTCTCATTGGGGTTGACGGTGGCTGATGTTGCT344                               (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 423 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 4E5                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GTAAGGCCACATGCTGCCATGGGCTGGGGATCTAAGGTGTCGGTTAAGGACTTAGCCACC60                CCCGCGGGGAAGATGGCCGTCCATGACCGGCTTCAGGAGATACTTGAAGGGACTCCGGTC120               CCCTTTACTCTTACTGTGAAAAAGGAGGTGTTCTTCAAAGACCGGAAGGAGGAGAAGGCC180               CCCCGCCTCATTGTGTTCCCCCCCCTGGACTTCCGGATAGCTGAAAAGCTCATCTTGGGA240               GACCCAGGCCGGGTAGCCAAGGCGGTGTTGGGGGGGGCCTACGCCTTCCAGTACACCCCA300               AATCAGCGAGTTAAGGAGATGCTCAAGCTATGGGAGTCTAAGAAGACCCCTTGCGCCATC360               TGTGTGGACGCCACCTGCTTCGACAGTAGCATAACTGAAGAGGACGTGGCTTTGGAGACA420               GAG423                                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 516 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 3E5                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TACAGCCTATTGTGACCAGGTGCGCACTCCGCTTGAATTGCAGGTTGGGTGCTTGGTGGG60                CAATGAACTTACCTTTGAATGTGACAAGTGTGAGGCTAGGCAAGAAACCTTGGCCTCCTT120               CTCTTACATTTGGTCTGGAGTGCCGCTGACTAGGGCCACGCCGGCCAAGCCTCCCGTGGT180               GAGGCCGGTTGGCTCTTTGTTAGTGGCCGACACTACTAAGGTGTATGTTACCAATCCAGA240               CAATGTGGGACGGAGGGTGGACAAGGTGACCTTCTGGCGTGCTCCTAGGGTTCATGATAA300               GTACCTCGTGGACTCTATTGAGCGCGCTAAGAGGGCCGCTCAAGCCTGCCTAAGCATGGG360               TTACACTTATGAGGAAGCAATAAGGACTGTAAGGCCACATGCTGCCATGGGCTGGGGATC420               TAAGGTGTCGGTTAAGGACTTAGCCACCCCCGCGGGGAAGATGGCCGTCCATGACCGGCT480               TCAGGAGATACTTGAAGGGACTCCGGTCCCCTTTAC516                                       (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 518 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 2E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GAATGGGCAACTCAAAGAACCAGTTTACTCTACCAAGCTGTGCCGGCACTATTGGATGGG60                GACTGTCCCTGTGAACATGCTGGGTTACGGTGAAACGTCGCCTCTCCTGGCCTCCGACAC120               CCCGAAGGTTGTGCCCTTCGGGACGTCTGGCTGGGCTGAGGTGGTGGTGACCACTACCCA180               CGTGGTAATCAGGAGGACCTCCGCCTATAAGCTGCTGCGCCAGCAAATCCTATCGGCTGC240               TGTAGCTGAGCCCTACTACGTCGACGGCATTCCGGTCTCATGGGACGCGGACGCTCGTGC300               GCCCGCCATGGTCTATGGCCCTGGGCAAAGTGTTACCATTGACGGGGAGCGCTACACCTT360               GCCTCATCAACTGAGGCTCAGGAATGTGGCACCCTCTGAGGTTTCATCCGAGGTGTCCAT420               TGACATTGGGACGGAGACTGAAGACTCAGAACTGACTGAGGCCGATCTGCCGCCGGCGGC480               TGCTGCTCTCCAAGCGATCGAGAATGCTGCGAGGATTC518                                     (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 268 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 1E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CTTACTGAGGCCGATCTGCCGCCGGCGGCTGCTGCTCTCCAAGCGATCGAGAATGCTGCG60                AGGATTCTTGAACCGCACATTGATGTCATCATGGAGGACTGCAGTACACCCTCTCTTTGT120               GGTAGTAGCCGAGAGATGCCTGTATGGGGAGAAGACATCCCCCGTACTCCATCGCCAGCA180               CTTATCTCGGTTACTGAGAGCAGCTCAGATGAGAAGACCCCGTCGGTGTCCTCCTCGCAG240               GAGGATACCCCGTCCTCTGACTCATTCG268                                               (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 781 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: INDIVIDUAL CLONE 4E5-20                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GTAAGGCCACATGCTGCCATGGGCTGGGGATCTAAGGTGTCGGTTAAGGACTTAGCCACC60                CCCGCGGGGAAGATGGCCGTCCATGACCGGCTTCAGGAGATACTTGAAGGGACTCCGGTC120               CCCTTTACTCTTACTGTGAAAAAGGAGGTGTTCTTCAAAGACCGGAAGGAGGAGAAGGCC180               CCCCGCCTCATTGTGTTCCCCCCCCTGGACTTCCGGATAGCTGAAAAGCTCATCTTGGGA240               GACCCAGGCCGGGTAGCCAAGGCGGTGTTGGGGGGGGCCTACGCCTTCCAGTACACCCCA300               AATCAGCGAGTTAAGGAGATGCTCAAGCTATGGGAGTCTAAGAAGACCCCTTGCGCCATC360               TGTGTGGACGCCACCTGCTTCGACAGTAGCATAACTGAAGAGGACGTGGCTTTGGAGACA420               GAGTTATACGCTCTGGCCTCTGACCATCCAGAATGGGTGCGGGCACCTGGGAAATACTAT480               GCCTCAGGCACCATGGTCACCCCGGAAGGGGTGCCCGTCGGTGAGAGGTATTGCAGATCC540               TCGGGTGTCCTAACAACTAGCGCGAGCAACTGCCTGACCTGCTACATCAAGGTGAAAGCT600               GCCTGTGAGAGAGTGGGGCTGAAAAATGTCTCTCTTCTCATAGCCGGCGATGACTGCTTG660               ATCATATGTGAGCGGCCAGTGTGCGACCCAAGCGACGCTTTGGGCAGAGCCCTAGCGAGC720               TATGGGTACGCGTGCGAGCCCTCATATCATGCATCATTGGACACGGCCCCCTTCTGCTCC780               A781                                                                          (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PROBE 470- 201-1-142R                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      TCGGTTACTGAGAGCAGCTCAGATGAG27                                                 (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PROBE 470-20- 1-152F                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      TCGGTTACTGAGAGCAGCTCAGATGAG27                                                 (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 570 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone 470EXP1                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..570                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GCTGTATGGTTCTGGATTTCCATCTCACACAGGCTAGCAACATCAGCC48                            AlaValTrpPheTrpIleSerIleSerHisArgLeuAlaThrSerAla                              151015                                                                        ACCGTCAACCCCAATGAGAAAAAGCGCGTGACGCTCTTTTCAACGCAG96                            ThrValAsnProAsnGluLysLysArgValThrLeuPheSerThrGln                              202530                                                                        CACGACATCTTGACGGTAAGCTTCCTGGTCGCGTCGCTCTGTGGAAAT144                           HisAspIleLeuThrValSerPheLeuValAlaSerLeuCysGlyAsn                              354045                                                                        AAGGCTTTTAATACGGAAAGAGCCACGTTGAAGACACTTTCCTCCCCT192                           LysAlaPheAsnThrGluArgAlaThrLeuLysThrLeuSerSerPro                              505560                                                                        TCGGCTGTCTCGGACTCTTGGATGACCTCGAATGAGTCAGAGGACGGG240                           SerAlaValSerAspSerTrpMetThrSerAsnGluSerGluAspGly                              65707580                                                                      GTATCCTCCTGCGAGGAGGACACCGACGGGGTCTTCTCATCTGAGCTG288                           ValSerSerCysGluGluAspThrAspGlyValPheSerSerGluLeu                              859095                                                                        CTCTCAGTAACCGAGATAAGTGCTGGCGATGGAGTACGGGGGATGTCT336                           LeuSerValThrGluIleSerAlaGlyAspGlyValArgGlyMetSer                              100105110                                                                     TCTCCCCATACAGGCATCTCTCGGCTACTACCACAAAGAGAGGGTGTA384                           SerProHisThrGlyIleSerArgLeuLeuProGlnArgGluGlyVal                              115120125                                                                     CTGCAGTCCTCCATGATGACATCAATGTGCGGTTCAAGAATCCTCGCA432                           LeuGlnSerSerMetMetThrSerMetCysGlySerArgIleLeuAla                              130135140                                                                     GCATTCTCGATCGCTTGGAGAGCAGCAGCCGCCGGCGGCAGATCGGCC480                           AlaPheSerIleAlaTrpArgAlaAlaAlaAlaGlyGlyArgSerAla                              145150155160                                                                  TCAGTCAGTTCTGAGTCTTCAGTCTCCGTCCCAATGTCAATGGACACC528                           SerValSerSerGluSerSerValSerValProMetSerMetAspThr                              165170175                                                                     TCGGATGAAACCTCAGAGGGTGCCACATTCCTGAGCCTCAGT570                                 SerAspGluThrSerGluGlyAlaThrPheLeuSerLeuSer                                    180185190                                                                     (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 190 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      AlaValTrpPheTrpIleSerIleSerHisArgLeuAlaThrSerAla                              151015                                                                        ThrValAsnProAsnGluLysLysArgValThrLeuPheSerThrGln                              202530                                                                        HisAspIleLeuThrValSerPheLeuValAlaSerLeuCysGlyAsn                              354045                                                                        LysAlaPheAsnThrGluArgAlaThrLeuLysThrLeuSerSerPro                              505560                                                                        SerAlaValSerAspSerTrpMetThrSerAsnGluSerGluAspGly                              65707580                                                                      ValSerSerCysGluGluAspThrAspGlyValPheSerSerGluLeu                              859095                                                                        LeuSerValThrGluIleSerAlaGlyAspGlyValArgGlyMetSer                              100105110                                                                     SerProHisThrGlyIleSerArgLeuLeuProGlnArgGluGlyVal                              115120125                                                                     LeuGlnSerSerMetMetThrSerMetCysGlySerArgIleLeuAla                              130135140                                                                     AlaPheSerIleAlaTrpArgAlaAlaAlaAlaGlyGlyArgSerAla                              145150155160                                                                  SerValSerSerGluSerSerValSerValProMetSerMetAspThr                              165170175                                                                     SerAspGluThrSerGluGlyAlaThrPheLeuSerLeuSer                                    180185190                                                                     (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1288 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 5E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      ACGGGTAGGGGCAGGTCTGGACGCTACTACTACGCGGGGGTGGGCAAAGCCCCTGCGGGT60                GTGGTGCGCTCAGGTCCTGTCTGGTCGGCGGTGGAAGCTGGAGTGACCTGGTACGGAATG120               GAACCTGACTTGACAGCTAACCTACTGAGACTTTACGACGACTGCCCTTACACCGCAGCC180               GTCGCGGCTGATATCGGAGAAGCCGCGGTGTTCTTCTCTGGGCTCGCCCCATTGAGGATG240               CACCCTGATGTCAGCTGGGCAAAAGTTCGCGGCGTCAACTGGCCCCTCTTGGTGGGTGTT300               CAGCGGACCATGTGTCGGGAAACACTGTCTCCCGGCCCATCGGATGACCCCCAATGGGCA360               GGTCTGAAGGGCCCAAATCCTGTCCCACTCCTGCTGAGGTGGGGCAATGATTTACCATCT420               AAAGTGGCCGGCCACCACATAGTGGACGACCTGGTCCGGAGACTCGGTGTGGCGGAGGGT480               TACGTCCGCTGCGACGCTGGGCCGATCTTGATGATCGGTCTAGCTATCGCGGGGGGAATG540               ATCTACGCGTCATACACCGGGTCGCTAGTGGTGGTGACAGACTGGGATGTGAAGGGGGGT600               GGCGCCCCCCTTTATCGGCATGGAGACCAGGCCACGCCTCAGCCGGTGGTGCAGGTTCCT660               CCGGTAGACCATCGGCCGGGGGGTGAATCAGCACCATCGGATGCCAAGACAGTGACAGAT720               GCGGTGGCAGCCATCCAGGTGGACTGCGATTGGACTATCATGACTCTGTCGATCGGAGAA780               GTGTTGTCCTTGGCTCAGGCTAAGACGGCCGAGGCCTACACAGCAACCGCCAAGTGGCTC840               GCTGGCTGCTATACGGGGACGCGGGCCGTTCCCACTGTATCCATTGTTGACAAGCTCTTC900               GCCGGAGGGTGGGCGGCTGTGGTGGGCCATTGCCACAGCGTGATTGCTGCGGCGGTGGCG960               GCCTACGGGGCTTCAAGGAGCCCGCCGTTGGCAGCCGCGGCTTCCTACCTGATGGGGTTG1020              GGCGTTGGAGGCAACGCTCAGACGCGCCTGGCGTCTGCCCTCCTATTGGGGGCTGCTGGA1080              ACCGCCTTGGGCACTCCTGTCGTGGGCTTGACCATGGCAGGTGCGTTCATGGGGGGGGCC1140              AGTGTCTCCCCCTCCTTGGTCACCATTTTATTGGGGGCCGTCGGAGGTTGGGAGGGTGTT1200              GTCAACGCGGCGAGCCTAGTCTTTGACTTCATGGCGGGGAAACTTTCATCAGAAGATCTG1260              TGGTATGCCATCCCGGTACTGACCAGCC1288                                              (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 862 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 6E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      ACGGCAACATGGGGCACAAGGTCTTAATCTTGAACCCCTCAGTGGCCACTGTGCGGGCCA60                TGGGCCCGTACATGGAGCGGCTGGCGGGTAAACATCCAAGTATATACTGTGGGCATGATA120               CAACTGCTTTCACAAGGATCACTGACTCCCCCCTGACGTATTCAACCTATGGGAGGTTTT180               TGGCCAACCCTAGGCAGATGCTACGGGGCGTTTCGGTGGTCATTTGTGATGAGTGCCACA240               GTCATGACTCAACCGTGCTGTTAGGCATTGGGAGAGTTCGGGAGCTGGCGCGTGGGTGCG300               GAGTGCAACTAGTGCTCTACGCCACCGCTACACCTCCCGGATCCCCTATGACGCAGCACC360               CTTCCATAATTGAGACAAAATTGGACGTGGGCGAGATTCCCTTTTATGGGCATGGAATAC420               CCCTCGAGCGGATGCGAACCGGAAGGCACCTCGTGTTCTGCCATTCTAAGGCTGAGTGCG480               AGCGCCTTGCTGGCCAGTTCTCCGCTAGGGGGGTCAATGCCATTGCCTATTATAGGGGTA540               AAGACAGTTCTATCATCAAGGATGGGGACCTGGTGGTCTGTGCTACAGACGCGCTTTCCA600               CTGGGTACACTGGAAATTTCGACTCCGTCACCGACTGTGGATTAGTGGTGGAGGAGGTCG660               TTGAGGTGACCCTTGATCCCACCATTACCATCTCCCTGCGGACAGTGCCTGCGTCGGCTG720               AACTGTCGATGCAAAGACGAGGACGCACGGGTAGGGGCAGGTCTGGACGCTACTACTACG780               CGGGGGTGGGCAAAGCCCCTGCGGGTGTGGTGCGCTCAGGTCCTGTCTGGTCGGCGGTGG840               AAGCTGGAGTGACCTCGTACGG862                                                     (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 865 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Individual Clone GE3L-11                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AGTACGGCAACATGGGGCACAAGGTCTTAATCTTGAACCCCTCAGTGGCCACTGTGCGGG60                CCATGGGCCCGTACATGGAGCGGCTGGCGGGTAAACATCCAAGTATATACTGTGGGCATG120               ATACAACTGCTTTCACAAGGATCACTGACTCCCCCCTGACGTATTCAACCTATGGGAGGT180               TTTTGGCCAACCCTAGGCAGATGCTACGGGGCGTTTCGGTGGTCATTTGTGATGAGTGCC240               ACAGTCATGACTCAACCGTGCTGTTAGGCATTGGGAGAGTCCGGGAGCTGGCGCGTGGGT300               GCGGGGTGCAACTAGTGCTCTACGCCACCGCTACACCTCCCGGATCCCCTATGACGCAGC360               ACCCTTCCATAATTGAGACAAAATTGGACGTGGGCGAGATTCCCTTTTATGGACATGGAA420               TACCCCTCGAGCGGATGCGAACCGGAAGGCACCTCGTGTTCTGCCATTCTAAGGCTGAGT480               GCGAGCGCCTTGCTGGCCAGTTCTCCGCTAGGGGGGTCAATGCCATTGCCTATTATAGGG540               GTAAAGACAGTTCTATCATCAAGGATGGGGACCTGGTGGTCTGTGCTACAGACGCGCTTT600               CCACTGGGTACACTGGAAATTTCGACTCCGTCACCGACTGTGGATTAGTGGTGGAGGAGG660               TCGTTGAGGTGACCCTTGATCCCACCATTACCATCTCCCTGCGGACAGTGCCTGCGTCGG720               CTGAACTGTCGATGCAAAGACGAGGACGCACGGGTAGGGGCAGGTCTGGACGCTACTACT780               ACGCGGGGGTGGGCAAAGCCCCTGCGGGTGTGGTGCGCTCAGGTCCTGTCTGGTCGGCGG840               TGGAAGCTGGAGTGACCTCGTACGG865                                                  (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 596 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 7E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AGCATGGGAACATGCTTGAACGGCCTGCTGTTCACGACCTTCCATGGGGCTTCATCCCGA60                ACCATCGCCACACCCGTGGGGGCCCTTAATCCCAGATGGTGGTCAGCCAGTGATGATGTC120               ACGGTGTATCCACTCCCGGATGGGGCTACTTCGTTAACACCTTGTACTTGCCAGGCTGAG180               TCCTGTTGGGTCATCAGATCCGACGGGGCCCTATGCCATGGCTTGAGCAAGGGGGACAAG240               GTGGAGCTGGATGTGGCCATGGAGGTCTCTGACTTCCGTGGCTCGTCTGGCTCACCGGTC300               CTATGTGACGAAGGGCACGCAGTAGGAATGCTCGTGTCTGTGCTTCACTCCGGTGGTAGG360               GTCACCGCGGCACGGTTCACTAGGCCGTGGACCCAAGTGCCAACAGATGCCAAAACCACT420               ACTGAACCCCCTCCGGTGCCGGCCAAAGGAGTTTTCAAAGAGGCCCCGTTGTTTATGCCT480               ACGGGAGCGGGAAAGAGCACTCGCGTCCCGTTGGAGTACGATAACATGGGGCACAAGGTC540               TTAATCTTGAACCCCTCAGTGGCCACTGTGCGGGCCATGGGCCCGTACATGGAGCG596                   (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 586 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 5E5                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GAGCTATGGGTACGCGTGCGAGCCCTCATATCATGCATCATTGGACACGGCCCCCTTCTG60                CTCCACTTGGCTTGCTGAGTGCAATGCAGATGGGAAGCGCCATTTCTTCCTGACCACGGA120               CTTCCGGAGGCCGCTCGCTCGCATGTCGAGTGAGTATAGTGACCCGATGGCTTCGGCGAT180               CGGTTACATCCTCCTTTATCCTTGGCACCCCATCACACGGTGGGTCATCATCCCTCATGT240               GCTAACGTGCGCATTCAGGGGTGGAGGCACACCGTCTGATCCGGTTTGGTGCCAGGTGCA300               TGGTAACTACTACAAGTTTCCACTGGACAAACTGCCTAACATCATCGTGGCCCTCCACGG360               ACCAGCAGCGTTGAGGGTTACCGCAGACACAACTAAAACAAAGATGGAGGCTGGTAAGGT420               TCTGAGCGACCTCAAGCTCCCTGGCTTAGCAGTCCACCGAAAGAAGGCCGGGGCGTTGCG480               AACACGCATGCTCCGCTCGCGCGGTTGGGCTGAGTTGGCTAGGGGCTTGTTGTGGCATCC540               AGGCCTACGGCTTCCTCCCCCTGAGATTGCTGGTATCCCGGGGGGT586                             (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 242 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 6E5 (44F)                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      CGAACGCGCATGCTCCGCTCGCGCGGTTGGGCTGAGTTGGCTAGGGGCTTGTTGTGGCAT60                CCAGGCCTACGGCTTCCTCCCCCTGAGATTGCTGGTATCCCGGGGGGTTTCCCTCTCTCC120               CCCCCCTATATGGGGGTGGTACACCAATTGGATTTCACAAGCCAGAGGAGTCGCTGGCGG180               TGGTTGGGGTTCTTAGCCCTGCTCATCGTAGCCCTCTTCGGGTGAACTAAATTCATCTGT240               TG242                                                                         (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer Gt11 rev-JL                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      TGGTAATGGTAGCGACCGGCGCTCAGC27                                                 (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE- 3F                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      GCCGCCATGGTCTCATGGGACGCGGACGCTCGTGCGCCCGCGATG45                               (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE- 3R                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GCGCGGATCCGATAAGTGCTGGCGATGGAGTACG34                                          (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE- 9F                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GGCACCATGGTCACCCCGGAAG22                                                      (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE- 9R                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      GCTCGGATCCGGAGCAGAAGGGGGCCGT28                                                (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 364 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: GE3-2                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..364                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GGTCTCATGGGACGCGGACGCTCGTGCGCCCGCGATGGTCTATGGC46                              ValSerTrpAspAlaAspAlaArgAlaProAlaMetValTyrGly                                 151015                                                                        CCTGGGCAAAGTGTTACCATTGACGGGGAGCGCTACACCTTGCCTCAT94                            ProGlyGlnSerValThrIleAspGlyGluArgTyrThrLeuProHis                              202530                                                                        CAACTGAGGCTCAGGAATGTGGCACCCTCTGAGGTTTCATCCGAGGTG142                           GlnLeuArgLeuArgAsnValAlaProSerGluValSerSerGluVal                              354045                                                                        TCCATTGACATTGGGACGGAGACTGAAGACTCAGAACTGACTGAGGCC190                           SerIleAspIleGlyThrGluThrGluAspSerGluLeuThrGluAla                              505560                                                                        GATCTGCCGCCGGCGGCTGCTGCTCTCCAAGCGATCGAGAATGCTGCG238                           AspLeuProProAlaAlaAlaAlaLeuGlnAlaIleGluAsnAlaAla                              657075                                                                        AGGATTCTTGAACCGCACATTGATGTCATCATGGAGGACTGCAGTACA286                           ArgIleLeuGluProHisIleAspValIleMetGluAspCysSerThr                              80859095                                                                      CCCTCTCTTTGTGGTAGTAGCCGAGAGATGCCTGTATGGGGAGAAGAC334                           ProSerLeuCysGlySerSerArgGluMetProValTrpGlyGluAsp                              100105110                                                                     ATCCCCCGTACTCCATCGCCAGCACTTATC364                                             IleProArgThrProSerProAlaLeuIle                                                115120                                                                        (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      ValSerTrpAspAlaAspAlaArgAlaProAlaMetValTyrGlyPro                              151015                                                                        GlyGlnSerValThrIleAspGlyGluArgTyrThrLeuProHisGln                              202530                                                                        LeuArgLeuArgAsnValAlaProSerGluValSerSerGluValSer                              354045                                                                        IleAspIleGlyThrGluThrGluAspSerGluLeuThrGluAlaAsp                              505560                                                                        LeuProProAlaAlaAlaAlaLeuGlnAlaIleGluAsnAlaAlaArg                              65707580                                                                      IleLeuGluProHisIleAspValIleMetGluAspCysSerThrPro                              859095                                                                        SerLeuCysGlySerSerArgGluMetProValTrpGlyGluAspIle                              100105110                                                                     ProArgThrProSerProAlaLeuIle                                                   115120                                                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 290 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone GE9- 2                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..290                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      CCATGGTCACCCCGGAAGGGGTGCCCGTTGGTGAGAGGTATTGCAGA47                             MetValThrProGluGlyValProValGlyGluArgTyrCysArg                                 151015                                                                        TCCTCGGGTGTCCTAACAACTAGCGCGAGCAACTGCTTGACCTGCTAC95                            SerSerGlyValLeuThrThrSerAlaSerAsnCysLeuThrCysTyr                              202530                                                                        ATCAAGGTGAAAGCCGCCTGTGAGAGGGTGGGGCTGAAAAATGTCTCT143                           IleLysValLysAlaAlaCysGluArgValGlyLeuLysAsnValSer                              354045                                                                        CTTCTCATAGCCGGCGATGACTGCTTGATCATATGTGAGCGGCCAGTG191                           LeuLeuIleAlaGlyAspAspCysLeuIleIleCysGluArgProVal                              505560                                                                        TGCGACCCAAGCGACGCTTTGGGCAGAGCCCTAGCGAGCTATGGGTAC239                           CysAspProSerAspAlaLeuGlyArgAlaLeuAlaSerTyrGlyTyr                              657075                                                                        GCGTGCGAGCCCTCATATTATGCATGCTCGGACACGGCCCCCTTCTGC287                           AlaCysGluProSerTyrTyrAlaCysSerAspThrAlaProPheCys                              80859095                                                                      TCC290                                                                        Ser                                                                           (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      MetValThrProGluGlyValProValGlyGluArgTyrCysArgSer                              151015                                                                        SerGlyValLeuThrThrSerAlaSerAsnCysLeuThrCysTyrIle                              202530                                                                        LysValLysAlaAlaCysGluArgValGlyLeuLysAsnValSerLeu                              354045                                                                        LeuIleAlaGlyAspAspCysLeuIleIleCysGluArgProValCys                              505560                                                                        AspProSerAspAlaLeuGlyArgAlaLeuAlaSerTyrGlyTyrAla                              65707580                                                                      CysGluProSerTyrTyrAlaCysSerAspThrAlaProPheCysSer                              859095                                                                        (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: JML-A SISPA Primer                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      AGGAATTCAGCGGCCGCGAG20                                                        (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: JML-B SISPA Primer                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      CTCGCGGCCGCTGAATTCCTTT22                                                      (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 470ep-f1 Primer                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GCGAATTCGCCATGGCGGGGAGACTTTCATCA32                                            (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 470ep-R1 Primer                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GCGAATTCGGATCCAGGGCCATAGACCATCGCGGG35                                         (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 470ep-f2 Primer                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      GCGAATTCCGTGCGCCCGCCATGGTC26                                                  (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 470ep-R3 Primer                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      GCGAATTCGGATCCCAAGGTTTCTTGCCTAGC32                                            (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 470ep-f4 Primer                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      GCGAATTCAAGTGTGAGGCTAGGCAA26                                                  (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 470ep-R4 Primer                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GCGAATTCGGATCCCCACACAGATGGCGCAAGGGG35                                         (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: KL-1 SISPA Primer                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GCAGGATCCGAATTCGCATCTAGAGAT27                                                 (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: KL-2 SISPA Primer                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      ATCTCTAGATGCGAATTCGGATCCTGCGA29                                               (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 186 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 10                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..186                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      CGTGCGCCCGCCATGGTCTATGGCCCTGGGCAAAGTGTTGCCATTGAC48                            ArgAlaProAlaMetValTyrGlyProGlyGlnSerValAlaIleAsp                              151015                                                                        GGGGAGCGCTACACCTTGCCTCATCAACTGAGGCTCAGGAATGTGGCA96                            GlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnValAla                              202530                                                                        CCCTCTGAGGTTTCATCCGAGGTGTCCATTGACATTGGGACGGAGGCT144                           ProSerGluValSerSerGluValSerIleAspIleGlyThrGluAla                              354045                                                                        GAAAACTCAGAACTGACTGAGGCCGATCTGCCGCCGGCGGCT186                                 GluAsnSerGluLeuThrGluAlaAspLeuProProAlaAla                                    505560                                                                        (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      ArgAlaProAlaMetValTyrGlyProGlyGlnSerValAlaIleAsp                              151015                                                                        GlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnValAla                              202530                                                                        ProSerGluValSerSerGluValSerIleAspIleGlyThrGluAla                              354045                                                                        GluAsnSerGluLeuThrGluAlaAspLeuProProAlaAla                                    505560                                                                        (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 282 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 12                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..282                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      CGTGCGCCCGCCATGGTCTATGGCCCTGGGCAAAGTGTTACCATTGAC48                            ArgAlaProAlaMetValTyrGlyProGlyGlnSerValThrIleAsp                              151015                                                                        GGGGAGCGCTACACCTTGCCTCATCAACTGAGGCTCAGGAATGTGGCA96                            GlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnValAla                              202530                                                                        CCCTCTGAGGTTTCATCCGAGGTGTCCATTGACATTGGGACGGAGACT144                           ProSerGluValSerSerGluValSerIleAspIleGlyThrGluThr                              354045                                                                        GAAGACTCAGAACTGACTGAGGCCGATCTGCCGCCGGCGGCTGCTGCT192                           GluAspSerGluLeuThrGluAlaAspLeuProProAlaAlaAlaAla                              505560                                                                        CTCCAAGCGATCGAGAATGCTGCGAGGATTCTTGAACCGCACATTGAT240                           LeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIleAsp                              65707580                                                                      GTCATCATGGAGGACTGCAGTACACCCTCTCTTTGTGGTAGT282                                 ValIleMetGluAspCysSerThrProSerLeuCysGlySer                                    8590                                                                          (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      ArgAlaProAlaMetValTyrGlyProGlyGlnSerValThrIleAsp                              151015                                                                        GlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnValAla                              202530                                                                        ProSerGluValSerSerGluValSerIleAspIleGlyThrGluThr                              354045                                                                        GluAspSerGluLeuThrGluAlaAspLeuProProAlaAlaAlaAla                              505560                                                                        LeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIleAsp                              65707580                                                                      ValIleMetGluAspCysSerThrProSerLeuCysGlySer                                    8590                                                                          (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 279 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 26                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..279                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      CGTGCGCCCGCCATGGTCTATGGCCCTGGGCAAAGTGTTTCCATTGAC48                            ArgAlaProAlaMetValTyrGlyProGlyGlnSerValSerIleAsp                              151015                                                                        GGGGAGCGCTACACCTTGCCTCATCAACTGAGGCTCAGGAATGTGGCA96                            GlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnValAla                              202530                                                                        CCCTCTGAGGTTTCATCCGAGGTGTCCATTGACATTGGGACGGAGACT144                           ProSerGluValSerSerGluValSerIleAspIleGlyThrGluThr                              354045                                                                        GAAGACTCAGAACTGACTGAGGCCGACCTGCCGCCGGCGGCTGCTGCT192                           GluAspSerGluLeuThrGluAlaAspLeuProProAlaAlaAlaAla                              505560                                                                        CTCCAAGCGATCGAGAATGCTGCGAGGATTCTTGAACCGCACATCGAT240                           LeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIleAsp                              65707580                                                                      GTCATCATGGAGGACTGCAGTACACCCTCTCTTTGTGGT279                                    ValIleMetGluAspCysSerThrProSerLeuCysGly                                       8590                                                                          (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      ArgAlaProAlaMetValTyrGlyProGlyGlnSerValSerIleAsp                              151015                                                                        GlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnValAla                              202530                                                                        ProSerGluValSerSerGluValSerIleAspIleGlyThrGluThr                              354045                                                                        GluAspSerGluLeuThrGluAlaAspLeuProProAlaAlaAlaAla                              505560                                                                        LeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIleAsp                              65707580                                                                      ValIleMetGluAspCysSerThrProSerLeuCysGly                                       8590                                                                          (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 5                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..108                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      GCCTATTGTGACAAGGTGCGCACTCCGCTTGAATTGCAGGTTGGGTGC48                            AlaTyrCysAspLysValArgThrProLeuGluLeuGlnValGlyCys                              151015                                                                        TTGGTGGGCAATGAACTTACCTTTGAATGTGACAAGTGTGAGGCTAGG96                            LeuValGlyAsnGluLeuThrPheGluCysAspLysCysGluAlaArg                              202530                                                                        CAAGAAACCTTG108                                                               GlnGluThrLeu                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      AlaTyrCysAspLysValArgThrProLeuGluLeuGlnValGlyCys                              151015                                                                        LeuValGlyAsnGluLeuThrPheGluCysAspLysCysGluAlaArg                              202530                                                                        GlnGluThrLeu                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 132 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 3                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..132                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      GAGATGGAAATCCAGAACCATACAGCCTATTGTGACAAGGTGCGCACT48                            GluMetGluIleGlnAsnHisThrAlaTyrCysAspLysValArgThr                              151015                                                                        CCGCTTGAATTGCAGGTTGGGTGCTTGGTGGGCAATGAACTTACCTTT96                            ProLeuGluLeuGlnValGlyCysLeuValGlyAsnGluLeuThrPhe                              202530                                                                        GAATGTGACAAGTGTGAGGCTAGGCAAGAAACCTTG132                                       GluCysAspLysCysGluAlaArgGlnGluThrLeu                                          3540                                                                          (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      GluMetGluIleGlnAsnHisThrAlaTyrCysAspLysValArgThr                              151015                                                                        ProLeuGluLeuGlnValGlyCysLeuValGlyAsnGluLeuThrPhe                              202530                                                                        GluCysAspLysCysGluAlaArgGlnGluThrLeu                                          3540                                                                          (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 258 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 27                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..258                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      AAAGCCTTATTTCCACAGAGCGACGCGACCAGGAAGCTTACCGTCAAG48                            LysAlaLeuPheProGlnSerAspAlaThrArgLysLeuThrValLys                              151015                                                                        ATGTCATGCTGCGTTGAAAAGAGCGTCACGCGCTTTTTCTCATTGGGG96                            MetSerCysCysValGluLysSerValThrArgPhePheSerLeuGly                              202530                                                                        TTGACGGTGGCTGATGTTGCTAGCCTGTGTGAGATGGAAATCCAGAAC144                           LeuThrValAlaAspValAlaSerLeuCysGluMetGluIleGlnAsn                              354045                                                                        CATATAGCCTATTGTGACAAGGTGCGCACTCCGCTTGAATTGCAGGTT192                           HisIleAlaTyrCysAspLysValArgThrProLeuGluLeuGlnVal                              505560                                                                        GGGTGCTTGGTGGGCAATGAACTCACCTTTGAATGTGACAAGTGTGAG240                           GlyCysLeuValGlyAsnGluLeuThrPheGluCysAspLysCysGlu                              65707580                                                                      GCTAGGCAAGAAACCTTG258                                                         AlaArgGlnGluThrLeu                                                            85                                                                            (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 86 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      LysAlaLeuPheProGlnSerAspAlaThrArgLysLeuThrValLys                              151015                                                                        MetSerCysCysValGluLysSerValThrArgPhePheSerLeuGly                              202530                                                                        LeuThrValAlaAspValAlaSerLeuCysGluMetGluIleGlnAsn                              354045                                                                        HisIleAlaTyrCysAspLysValArgThrProLeuGluLeuGlnVal                              505560                                                                        GlyCysLeuValGlyAsnGluLeuThrPheGluCysAspLysCysGlu                              65707580                                                                      AlaArgGlnGluThrLeu                                                            85                                                                            (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 25                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..108                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      ACCTATTGTGACAAGGTGCGCACTCCGCTTGAATTGCAGGTTGGGTGC48                            ThrTyrCysAspLysValArgThrProLeuGluLeuGlnValGlyCys                              151015                                                                        TTGGTGGGCAATGAACTTACCTTTGAATGTGACAAGTGTGAGGCTAGG96                            LeuValGlyAsnGluLeuThrPheGluCysAspLysCysGluAlaArg                              202530                                                                        CAAGAAACCTTG108                                                               GlnGluThrLeu                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      ThrTyrCysAspLysValArgThrProLeuGluLeuGlnValGlyCys                              151015                                                                        LeuValGlyAsnGluLeuThrPheGluCysAspLysCysGluAlaArg                              202530                                                                        GlnGluThrLeu                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 20                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 52..108                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      GCCGACACTACTAAGGTGTATGTTACCAATCCAGACAATGTGGGACGAAGGGTGGGC57                   ValGly                                                                        AATGAACTTACCTTTGAATGTGACAAGTGTGAGGCTAGGCAAGAAACC105                           AsnGluLeuThrPheGluCysAspLysCysGluAlaArgGlnGluThr                              51015                                                                         TTG108                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      ValGlyAsnGluLeuThrPheGluCysAspLysCysGluAlaArgGln                              151015                                                                        GluThrLeu                                                                     (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 168 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 16                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..168                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      TTGGGGTTGACGGTGGCTGATGTTGCTAGCCTGTGTGAGATGGAAATC48                            LeuGlyLeuThrValAlaAspValAlaSerLeuCysGluMetGluIle                              151015                                                                        CAGAACCATACAGCCTATTGTGACAAGGTGCGCACTCCGCTTGAATTG96                            GlnAsnHisThrAlaTyrCysAspLysValArgThrProLeuGluLeu                              202530                                                                        CAGGTTGGGTGCTTGGTGGGCAATGAACTTACCTTTGAATGTGACAAG144                           GlnValGlyCysLeuValGlyAsnGluLeuThrPheGluCysAspLys                              354045                                                                        TGTGAGGCTAGGCAAGAAACCTTG168                                                   CysGluAlaArgGlnGluThrLeu                                                      5055                                                                          (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      LeuGlyLeuThrValAlaAspValAlaSerLeuCysGluMetGluIle                              151015                                                                        GlnAsnHisThrAlaTyrCysAspLysValArgThrProLeuGluLeu                              202530                                                                        GlnValGlyCysLeuValGlyAsnGluLeuThrPheGluCysAspLys                              354045                                                                        CysGluAlaArgGlnGluThrLeu                                                      5055                                                                          (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 313 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 50                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..313                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      ATCACCGTCAACCCCAATGAGAAAAAGCGCGTGACGCTCTTTTCAACG48                            IleThrValAsnProAsnGluLysLysArgValThrLeuPheSerThr                              151015                                                                        CAGCACGACATCTTGACGGTAAGCTTCCTGGTCGCGTCGCTCTGTGGA96                            GlnHisAspIleLeuThrValSerPheLeuValAlaSerLeuCysGly                              202530                                                                        AATAAGGCTTTTAATACGGAAAGAGCCACGTTGAAGACACTTTCCTCC144                           AsnLysAlaPheAsnThrGluArgAlaThrLeuLysThrLeuSerSer                              354045                                                                        CCTTCGGCTGTCTCGGACTCTTGGATGACCTCGAATGAGTCAGAGGAC192                           ProSerAlaValSerAspSerTrpMetThrSerAsnGluSerGluAsp                              505560                                                                        GGGGTATCCTCCTGCGAGGAGGACACCGACGGGGTCTTCTCATCTGAG240                           GlyValSerSerCysGluGluAspThrAspGlyValPheSerSerGlu                              65707580                                                                      CTGCTCTCAGTAACCGAGATAAGTGCTGGCGATGGAGTACGGGGGATG288                           LeuLeuSerValThrGluIleSerAlaGlyAspGlyValArgGlyMet                              859095                                                                        TCTTCTCCCCATACAGGCATCTCTC313                                                  SerSerProHisThrGlyIleSer                                                      100                                                                           (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 104 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      IleThrValAsnProAsnGluLysLysArgValThrLeuPheSerThr                              151015                                                                        GlnHisAspIleLeuThrValSerPheLeuValAlaSerLeuCysGly                              202530                                                                        AsnLysAlaPheAsnThrGluArgAlaThrLeuLysThrLeuSerSer                              354045                                                                        ProSerAlaValSerAspSerTrpMetThrSerAsnGluSerGluAsp                              505560                                                                        GlyValSerSerCysGluGluAspThrAspGlyValPheSerSerGlu                              65707580                                                                      LeuLeuSerValThrGluIleSerAlaGlyAspGlyValArgGlyMet                              859095                                                                        SerSerProHisThrGlyIleSer                                                      100                                                                           (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 89 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 52                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 28..87                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      ACTGAGAGCAGCTCAGATGAGAAGACCCCTTCGGCTGTCTCGGACTCTTGG51                         ProSerAlaValSerAspSerTrp                                                      15                                                                            ATGACCTCGAATGAGTCAGAGGACGGGGTATCCTCGCA89                                      MetThrSerAsnGluSerGluAspGlyValSerSer                                          101520                                                                        (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      ProSerAlaValSerAspSerTrpMetThrSerAsnGluSerGluAsp                              151015                                                                        GlyValSerSer                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 214 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 53                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..100                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      AATAAGGCTTTTAATACGGAAAGAGCCACGTTGAAGACACTTTCCTCC48                            AsnLysAlaPheAsnThrGluArgAlaThrLeuLysThrLeuSerSer                              151015                                                                        CCTTCGGCTGTCTCGGACTCTTGGATGACCTCGAATGAGTCAGAGGAC96                            ProSerAlaValSerAspSerTrpMetThrSerAsnGluSerGluAsp                              202530                                                                        GGGGATCTCTAGATGCGAATTCAAGTGTGAGGCTAGGCAAGAAACCTTGGCCTC150                     Gly                                                                           CTTCTCTTACATTTGGTCTGGAGTGCCGCTGACTAGGGCCACGCCGGCCAAGCCTCCCGT210               GGTG214                                                                       (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      AsnLysAlaPheAsnThrGluArgAlaThrLeuLysThrLeuSerSer                              151015                                                                        ProSerAlaValSerAspSerTrpMetThrSerAsnGluSerGluAsp                              202530                                                                        Gly                                                                           (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 55                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 52..113                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      CCATCGCCAGCACTTATCTCGGTTACTGAGAGCAGCTCAGATCAGAAGACCCCTTCG57                   ProSer                                                                        1                                                                             GCTGTCTCGGACTCTTGGATGACCTCGAATGAGTCAGAGGACGGGGTA105                           AlaValSerAspSerTrpMetThrSerAsnGluSerGluAspGlyVal                              51015                                                                         TCCTCGCA113                                                                   SerSer                                                                        20                                                                            (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      ProSerAlaValSerAspSerTrpMetThrSerAsnGluSerGluAsp                              151015                                                                        GlyValSerSer                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 330 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 56                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..330                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      ACGTTGAAGACACTTTCCTCCCCTTCGGCTGTCTCGGACTCTTGGATG48                            ThrLeuLysThrLeuSerSerProSerAlaValSerAspSerTrpMet                              151015                                                                        ACCTCGAATGAGTCAGAGGACGGGGTATCCTCCTGCGAGGAGGACACC96                            ThrSerAsnGluSerGluAspGlyValSerSerCysGluGluAspThr                              202530                                                                        GACGGGGTCTTCTCATCTGAGCTGCTCTCAGTAACCGAGATAAGTGCT144                           AspGlyValPheSerSerGluLeuLeuSerValThrGluIleSerAla                              354045                                                                        GGCGATGGAGTACGGGGGATGTCTTCTCCCCATACAGGCATCTCTCGG192                           GlyAspGlyValArgGlyMetSerSerProHisThrGlyIleSerArg                              505560                                                                        CTACTACCACAAAGAGAGGGTGTACTGCAGTCCTCCATGATGACATCA240                           LeuLeuProGlnArgGluGlyValLeuGlnSerSerMetMetThrSer                              65707580                                                                      ATGTGCGGTTCAAGAATCCTCGCAGCATTCTCGATCGCTTGGAGAGCA288                           MetCysGlySerArgIleLeuAlaAlaPheSerIleAlaTrpArgAla                              859095                                                                        GCAGCCGCCGGCGGCAGATCGGCCTCAGTCAGTTCTGAGTCT330                                 AlaAlaAlaGlyGlyArgSerAlaSerValSerSerGluSer                                    100105110                                                                     (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                      ThrLeuLysThrLeuSerSerProSerAlaValSerAspSerTrpMet                              151015                                                                        ThrSerAsnGluSerGluAspGlyValSerSerCysGluGluAspThr                              202530                                                                        AspGlyValPheSerSerGluLeuLeuSerValThrGluIleSerAla                              354045                                                                        GlyAspGlyValArgGlyMetSerSerProHisThrGlyIleSerArg                              505560                                                                        LeuLeuProGlnArgGluGlyValLeuGlnSerSerMetMetThrSer                              65707580                                                                      MetCysGlySerArgIleLeuAlaAlaPheSerIleAlaTrpArgAla                              859095                                                                        AlaAlaAlaGlyGlyArgSerAlaSerValSerSerGluSer                                    100105110                                                                     (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 195 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 57                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..195                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      ACGGAAAGAGCCACGTTGAAGACACTTTCCTCCCCTTCGGCTGCCTCG48                            ThrGluArgAlaThrLeuLysThrLeuSerSerProSerAlaAlaSer                              151015                                                                        GACTCTTGGATGACCTCGAATGAGTCGGAGGACGGGGTATCCTCCTGC96                            AspSerTrpMetThrSerAsnGluSerGluAspGlyValSerSerCys                              202530                                                                        GAAGAGGACACCGACGGGGTCTTCTCATCTGAGCTGCTCTCAGTAACC144                           GluGluAspThrAspGlyValPheSerSerGluLeuLeuSerValThr                              354045                                                                        GAGATAAGTGCTGGCGGTGGAGTACGGGGGATGTCTTCTCCCCATACG192                           GluIleSerAlaGlyGlyGlyValArgGlyMetSerSerProHisThr                              505560                                                                        GGC195                                                                        Gly                                                                           65                                                                            (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      ThrGluArgAlaThrLeuLysThrLeuSerSerProSerAlaAlaSer                              151015                                                                        AspSerTrpMetThrSerAsnGluSerGluAspGlyValSerSerCys                              202530                                                                        GluGluAspThrAspGlyValPheSerSerGluLeuLeuSerValThr                              354045                                                                        GluIleSerAlaGlyGlyGlyValArgGlyMetSerSerProHisThr                              505560                                                                        Gly                                                                           65                                                                            (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 60                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..115                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      AAGACACTTTCCTCCCCTTCGGCTGTCTCGGACTCTTGGATGACCTCG48                            LysThrLeuSerSerProSerAlaValSerAspSerTrpMetThrSer                              151015                                                                        AATGAGTCAGAGGACGGGGTATCCTCCTGCGAGGAGGACACCGACTGG96                            AsnGluSerGluAspGlyValSerSerCysGluGluAspThrAspTrp                              202530                                                                        GTCTTCTCATCTGAGCTGC115                                                        ValPheSerSerGluLeu                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:95:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                      LysThrLeuSerSerProSerAlaValSerAspSerTrpMetThrSer                              151015                                                                        AsnGluSerGluAspGlyValSerSerCysGluGluAspThrAspTrp                              202530                                                                        ValPheSerSerGluLeu                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:96:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone Y5- 63                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 19..93                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                      GAGAGCAGCTCAGATGAGAAGACACTTTCCTCCCCTTCGGCTGTCTCGGAC51                         LysThrLeuSerSerProSerAlaValSerAsp                                             1510                                                                          TCTTGGATGACCTCGAATGAGTCAGAGGACGGGGTATCCTCG93                                  SerTrpMetThrSerAsnGluSerGluAspGlyValSerSer                                    152025                                                                        (2) INFORMATION FOR SEQ ID NO:97:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                      LysThrLeuSerSerProSerAlaValSerAspSerTrpMetThrSer                              151015                                                                        AsnGluSerGluAspGlyValSerSer                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:98:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1181 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 8E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                      GCTGGCTGAGGCACGGTTGGTCCCGCTGATCTTGCTGCTGCTATGGTGGTGGGTGAACCA60                GCTGGCAGTCCTAGGGCTGCCGGCTGTGGAAGCCGCCGTGGCAGGTGAGGTCTTCGCGGG120               CCCTGCCCTGTCCTGGTGTCTGGGACTCCCGGTCGTCAGTATGATATTGGGTTTGGCAAA180               CCTGGTGCTGTACTTTAGATGGTTGGGACCCCAACGCCTGATGTTCCTCGTGTTGTGGAA240               GCTTGCTCGGGGAGCTTTCCCGCTGGCCCTCTTGATGGGGATTTCGGCGACCCGCGGGCG300               CACCTCAGTGCTCGGGGCCGAGTTCTGCTTCGATGCTACATTCGAGGTGGACACTTCGGT360               GTTGGGCTGGGTGGTGGCCAGTGTGGTAGCTTGGGCCATTGCGCTCCTGAGCTCGATGAG420               CGCAGGGGGGTGGAGGCACAAAGCCGTGATCTATAGGACGTGGTGTAAGGGGTACCAGGC480               AATCCGTCAAAGGGTGGTGAGGAGCCCCCTCGGGGAGGGGCGGCCTGCCAAACCCCTGAC540               CTTTGCCTGGTGCTTGGCCTCGTACATCTGGCCAGATGCTGTGATGATGGTGGTGGTTGC600               CTTGGTCCTTCTCTTTGGCCTGTTCGACGCGTTGGATTGGGCCTTGGAGGAGATCTTGGT660               GTCCCGGCCCTCGTTGCGGCGTTTGGCTCGGGTGGTTGAGTGCTGTGTGATGGCGGGTGA720               GAAGGCCACAACCGTCCGGCTGGTCTCCAAGATGTGTGCGAGAGGAGCTTATTTGTTCGA780               TCATATGGGCTCTTTTTCGCGTGCTGTCAAGGAGCGCCTGTTGGAATGGGACGCAGCTCT840               TGAACCTCTGTCATTCACTAGGACGGACTGTCGCATCATACGGGATGCCGCGAGGACTTT900               GTCCTGCGGGCAGTGCGTCATGGGTTTACCCGTGGTTGCGCGCCGTGGTGATGAGGTTCT960               CATCGGCGTCTTCCAGGATGTGAATCATTTGCCTCCCGGGTTTGTTCCGACCGCGCCTGT1020              TGTCATCCGACGGTGCGGAAAGGGCTTCTTGGGGGTCACAAAGGCTGCCTTGACAGGTCG1080              GGATCCTGACTTACATCCAGGGAACGTCATGGTGTTGGGGACGGCTACGTCGCGAAGCAT1140              GGGAACATGCTTGAACGGCCTGCTGTTCACGACCTTCCATG1181                                 (2) INFORMATION FOR SEQ ID NO:99:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer Y5-10- F1                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                      TCAGCCATGGCTCGTGCGCCCGCGATGGTC30                                              (2) INFORMATION FOR SEQ ID NO:100:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer Y5-10- R1                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                                     CGAGGATCCAGCCGCCGGCGGCAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:101:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer Y5- 16F1                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                                     GATTCCATGGGTTTGGGGTTGACGGTGGCTGA32                                            (2) INFORMATION FOR SEQ ID NO:102:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470EP- R3                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                                     GCGAATTCGGATCCCAAGGTTTCTTGCCTAGC32                                            (2) INFORMATION FOR SEQ ID NO:103:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer Y5-5- F1                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                                     GAGGCCATGGCCTATTGTGACAAGGTG27                                                 (2) INFORMATION FOR SEQ ID NO:104:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer PGEX- R                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                                     GACCGTCTCCGGGAGCT17                                                           (2) INFORMATION FOR SEQ ID NO:105:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 326 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone GE15                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..326                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                                     CCATGGAGGTCTCTGACTTCCGTGGCTCGTCTGGCTCACCGGTCCTA47                             MetGluValSerAspPheArgGlySerSerGlySerProValLeu                                 151015                                                                        TGTGACGAAGGGCACGCAGTAGGAATGCTCGTGTCTGTGCTTCACTCC95                            CysAspGluGlyHisAlaValGlyMetLeuValSerValLeuHisSer                              202530                                                                        GGTGGTAGGGTCACCGCGGCACGGTTCACTAGGCCGTGGACCCAAGTG143                           GlyGlyArgValThrAlaAlaArgPheThrArgProTrpThrGlnVal                              354045                                                                        CCAACAGATGCCAAAACCACCACTGAACCCCCTCCGGTGCCGGCCAAA191                           ProThrAspAlaLysThrThrThrGluProProProValProAlaLys                              505560                                                                        GGAGTTTTCAAAGAGGCCCCGTTGTTTATGCCTACGGGAGCGGGAAAG239                           GlyValPheLysGluAlaProLeuPheMetProThrGlyAlaGlyLys                              657075                                                                        AGCACTCGCGTCCCGTTGGAGTACGGCAACATGGGGCACAAGGTCTTA287                           SerThrArgValProLeuGluTyrGlyAsnMetGlyHisLysValLeu                              80859095                                                                      ATCTTGAACCCCTCAGTGGCCACTGTGCGGGCGATGGGC326                                    IleLeuAsnProSerValAlaThrValArgAlaMetGly                                       100105                                                                        (2) INFORMATION FOR SEQ ID NO:106:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                                     MetGluValSerAspPheArgGlySerSerGlySerProValLeuCys                              151015                                                                        AspGluGlyHisAlaValGlyMetLeuValSerValLeuHisSerGly                              202530                                                                        GlyArgValThrAlaAlaArgPheThrArgProTrpThrGlnValPro                              354045                                                                        ThrAspAlaLysThrThrThrGluProProProValProAlaLysGly                              505560                                                                        ValPheLysGluAlaProLeuPheMetProThrGlyAlaGlyLysSer                              65707580                                                                      ThrArgValProLeuGluTyrGlyAsnMetGlyHisLysValLeuIle                              859095                                                                        LeuAsnProSerValAlaThrValArgAlaMetGly                                          100105                                                                        (2) INFORMATION FOR SEQ ID NO:107:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 138 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Clone GE17                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..138                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                                     GGTGATGAGGTTCTCATCGGCGTCTTCCAGGATGTGAATCATTTGCCT48                            GlyAspGluValLeuIleGlyValPheGlnAspValAsnHisLeuPro                              151015                                                                        CCCGGGTTTGTTCCGACCGCGCCTGTTGTCATCCGACGGTGCGGAAAG96                            ProGlyPheValProThrAlaProValValIleArgArgCysGlyLys                              202530                                                                        GGCTTCTTGGGGGTCACAAAGGCTGCCTTGACAGGTCGGGAT138                                 GlyPheLeuGlyValThrLysAlaAlaLeuThrGlyArgAsp                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:108:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                                     GlyAspGluValLeuIleGlyValPheGlnAspValAsnHisLeuPro                              151015                                                                        ProGlyPheValProThrAlaProValValIleArgArgCysGlyLys                              202530                                                                        GlyPheLeuGlyValThrLysAlaAlaLeuThrGlyArgAsp                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:109:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 395 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 9E3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                                     TGTATTTGTCCTGTTATACCTGATGAAGCTGGCTGAGGCACGGTTGGTCCCGCTGATCTT60                GCTGCTGCTATGGTGGTGGGTGAACCAGCTGGCAGTCCTAGGGCTGCCGGCTGTGGAAGC120               CGCCGTGGCAGGTGAGGTCTTCGCGGGCCCTGCCCTGTCCTGGTGTCTGGGACTCCCGGT180               CGTCAGTATGATATTGGGTTTGGCAAACCTAGTGCTGTACTTTAGATGGTTGGGACCCCA240               ACGCCTGATGTTCCTCGTGTTGTGGAAGCTTGCTCGGGGAGCTTTCCCGCTGGCCCTCTT300               GATGGGGATTTCGGCGACCCGCGGGCGCACCTCAGTGCTCGGGGCCGAGTTCTGCTTCGA360               TGCTACATTCGAGGTGGACACTTCGGTGTTGGGCT395                                        (2) INFORMATION FOR SEQ ID NO:110:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 460 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 10E3                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                                     GCCCCTGGGCAACCAGGGCCGAGGCAACCCGGTGCGGTCGCCCTTGGGTTTTGGGTCCTA60                CGCCATGACCAGGATCCGAGATACCCTACATCTGGTGGAGTGTCCCACACCAGCCATTGA120               GCCTCCCACCGGGACGTTTGGGTTCTTCCCCGGGACGCCGCCTCTCAACAACTGCATGCT180               CTTGGGCACGGAAGTGTCCGAGGCACTTGGGGGGGCTGGCCTCACGGGGGGGTTCTATGA240               ACCCCTGGTGCGCAGGTGTTCGAAGCTGATGGGAAGCCGAAATCCGGTTTGTCCGGGGTT300               TGCATGGCTCTCTTCGGGCAGGCCTGATGGGTTTATACATGTCCAGGGTCACTTGCAGGA360               GGTGGATGCAGGCAACTTCATCCCGCCCCCGCGCTGGTTGCTCTTGGACTTTGTATTTGT420               CCTGTTATACCTGATGAAGCTGGCTGAGGCACGGTTGGTC460                                   (2) INFORMATION FOR SEQ ID NO:111:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE15F                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                                     GCCGCCATGGAGGTCTCTGACTTCCGTG28                                                (2) INFORMATION FOR SEQ ID NO:112:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE15R                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                                     GCGCGGATCCGCCCATCGCCCGCACAGTGGC31                                             (2) INFORMATION FOR SEQ ID NO:113:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE17F                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                                     CGCTCCATGGGTGATGAGGTTCTCATCGGCG31                                             (2) INFORMATION FOR SEQ ID NO:114:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE17R                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                                     GTAAGTCAGGATCCCGACCTGTCAAGGC28                                                (2) INFORMATION FOR SEQ ID NO:115:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 452 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: NcoI/EcoRI- containing fragment of                    pGEX-HISb- GE3-s HGV plasmid                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                                     CAAAATCGGATCTGGTTCCGCGTGGTTCCATGGTCTCATGGGACGCGGACGCTCGTGCGC60                CCGCGATGGTCTATGGCCCTGGGCAAAGTGTTACCATTGACGGGGAGCGCTACACCTTGC120               CTCATCAACTGAGGCTCAGGAATGTGGCACCCTCTGAGGTTTCATCCGAGGTGTCCATTG180               ACATTGGGACGGAGACTGAAGACTCAGAACTGACTGAGGCCGATCTGCCGCCGGCGGCTG240               CTGCTCTCCAAGCGATCGAGAATGCTGCGAGGATTCTTGAACCGCACATTGATGTCATCA300               TGGAGGACTGCAGTACACCCTCTCTTTGTGGTAGTAGCCGAGAGATGCCTGTATGGGGAG360               AAGACATCCCCCGTACTCCATCGCCAGCACTTATCGGATCCCACCATCACCATCACCATT420               AGAATTCATCGTGACTGACTGACGATCTACCT452                                           (2) INFORMATION FOR SEQ ID NO:116:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 590 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 11E3                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                                     AGCAATCGGCTGGGGTGACCCCATCACTTATTGGAGCCACGGGCAAAATCAGTGGCCCCT60                TTCATGCCCCCAGTATGTCTATGGGTCTGCTACAGTCACTTGCGTGTGGGGTTCCGCTTC120               TTGGTTTGCCTCCACCAGTGGTCGCGACTCGAAGATAGATGTGTGGAGTTTAGTGCCAGT180               TGGCTCTGCCACCTGCACCATAGCCGCACTTGGATCATCGGATCGCGACACGGTGCCTGG240               GCTCTCCGAGTGGGGAATCCCGTGCGTGACGTGTGTTCTGGACCGTCGGCCTGCCTCCTG300               CGGCACCTGTGTGAGGGACTGCTGGCCCGAGACCGGGTCGGTTAGGTTCCCATTCCATCG360               GTGCGGCGTGGGGCCTCGGCTGACAAAGGACTTGGAAGCTGTGCCCTTCGTCAACAGGAC420               AACTCCCTTCACCATTAGGGGGCCCCTGGGCAACCAGGGCCGAGGCAACCCGGTGCGGTC480               GCCCTTGGGTTTTGGGTCCTACGCCATGACCAGGATCCGAGATACCCTACATCTGGTGGA540               GTGTCCCACACCAGCCATCGAGCCTCCCACCGGGACGTTTGGGTTCTTCC590                         (2) INFORMATION FOR SEQ ID NO:117:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Probe E3- 111PROB                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                                     TGGTGAAGGGAGTTGTCCTATTGACGAAG29                                               (2) INFORMATION FOR SEQ ID NO:118:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 735 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 12E3                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                                     ATTGTTGTGCCCCGGAGGACATCGGGTTCTGCCTGGAGGGTGGATGCCTGGTGGCCCTGG60                GGTGCACGATTTGCACTGACCAATGCTGGCCACTGTATCAGGCGGGTTTGGCTGTGCGGC120               CTGGCAAGTCCGCGGCCCAACTGGTGGGGGAGCTGGGTAGCCTATACGGGCCCCTGTCGG180               TCTCGGCCTATGTGGCTGGGATCCTGGGCCTGGGTGAGGTGTACTCGGGTGTCCTAACGG240               TGGGAGTCGCGTTGACGCGCCGGGTCTACCCGGTGCCTAACCTGACGTGTGCAGTCGCGT300               GTGAGCTAAAGTGGGAAAGTGAGTTTTGGAGATGGACTGAACAGCTGGCCTCCAACTACT360               GGATTCTGGAATACCTCTGGAAGGTCCCATTTGATTTCTGGAGAGGCGTGATAAGCCTGA420               CCCCCTTGTTGGTTTGCGTGGCCGCATTGCTGCTGCTTGAGCAACGGATTGTCATGGTCT480               TCCTGTTGGTGACGATGGCCGGGATGTCGCAAGGCGCCCCTGCCTCCGTTTTGGGGTCAC540               GCCCCTTTGACTACGGGTTGACTTGGCAGACCTGCTCTTGCAGGGCCAACGGTTCGCGTT600               TTTCGACTGGGGAGAAGGTGTGGGACCGTGGGAACGTTACGCTTCAGTGTGACTGCCCTA660               ACGGCCCCTGGGTGTGGTTGCCAGCCTTTTGCCAAGCAATCGGCTGGGGTGACCCCATCA720               CTTATTGGAGCCACG735                                                            (2) INFORMATION FOR SEQ ID NO:119:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470EXT4-2189R                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                                     ATCTGTGGTATGCCATCCCGGT22                                                      (2) INFORMATION FOR SEQ ID NO:120:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470EXT4-29F                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:                                     GTTATGCTACTGTCGAAGCAGGT23                                                     (2) INFORMATION FOR SEQ ID NO:121:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: NS5 Primer GV57-4512 MF                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:                                     GGACTTCCGGATAGCTGARAAGCT24                                                    (2) INFORMATION FOR SEQ ID NO:122:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: NS5 Primer GV57-4657 MR                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:                                     GCRTCCACACAGATGGCGCA20                                                        (2) INFORMATION FOR SEQ ID NO:123:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: NS5 Probe GV22dc-89 MF                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:                                     CYCGCTGRTTTGGGGTGTACTGGAAGGC28                                                (2) INFORMATION FOR SEQ ID NO:124:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 5'-UTR Primer FV94-22F                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:                                     GAAAGCCCCAGAAACCGACGCCTATCTAAGT31                                             (2) INFORMATION FOR SEQ ID NO:125:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 5'UTR Primer FV94-724R                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:                                     GCACAGCCAAACCCGCCTGATACAGT26                                                  (2) INFORMATION FOR SEQ ID NO:126:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 5'-UTR Primer FV94-94F                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:                                     GTGGTGGATGGGTGATGACAGGGTTGGT28                                                (2) INFORMATION FOR SEQ ID NO:127:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 5'-UTR Primer FV94-912R                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:                                     TAACTCACACGCGACTGCACACGTCAGGT29                                               (2) INFORMATION FOR SEQ ID NO:128:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: ENV Library Primer GEP-F15                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:                                     GCGGCCATGGTGCCCTTCGTCAATAGGACA30                                              (2) INFORMATION FOR SEQ ID NO:129:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: ENV Library Primer GEP-R15                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:                                     CTTGCCATGGCCAGCTGGTTCACCCACCA29                                               (2) INFORMATION FOR SEQ ID NO:130:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- F17                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:                                     GCAGGATCCCCTCTGGAAGGTCCCATTTGA30                                              (2) INFORMATION FOR SEQ ID NO:131:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- R16                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:                                     TGCGAATCCTCGGCCCTGGTTGCCCAG27                                                 (2) INFORMATION FOR SEQ ID NO:132:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470ep- F9                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:                                     GCTAGATCTGGCAACATGGGGCACAAGGTC30                                              (2) INFORMATION FOR SEQ ID NO:133:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470ep- R9                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:                                     CACAGATCTCGCGTAGTAGTAGCGTCCAGA30                                              (2) INFORMATION FOR SEQ ID NO:134:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: AP Primer for Race PCR                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:                                     CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG38                                      (2) INFORMATION FOR SEQ ID NO:135:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- F10                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:                                     GCTGGATCCAGCATGGGAACATGCTTGAAC30                                              (2) INFORMATION FOR SEQ ID NO:136:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- R10                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:                                     CGCGGATCCCACAGTGGCCACTGAGGGGTT30                                              (2) INFORMATION FOR SEQ ID NO:137:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer EXY10- F1                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:                                     GCCCATATGGTGATCACTGGTGACGTT27                                                 (2) INFORMATION FOR SEQ ID NO:138:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer EXY10- F2                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:                                     GCCCATATGCTGGGTTACGGTGAA24                                                    (2) INFORMATION FOR SEQ ID NO:139:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer EXY10- F3                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:                                     GCCCATATGACCTCCGCCTATAAGCTG27                                                 (2) INFORMATION FOR SEQ ID NO:140:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer EXY10- R1                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:                                     GCCCATATGAGCCGCCGGCGGCAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:141:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer EXY5- R1                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:                                     TGCGGATCCCACATTGTCTGGATT24                                                    (2) INFORMATION FOR SEQ ID NO:142:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer Y5-5- F1                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:                                     TCGGCCATGGCCTATTGTGACAAGGTG27                                                 (2) INFORMATION FOR SEQ ID NO:143:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 219 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Antigen Clone Q7-12-1                                 (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..219                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:                                     GTGCCCTTCGTCAATAGGACAACTCTCTTCACCATTAGGGGGCCCCTG48                            ValProPheValAsnArgThrThrLeuPheThrIleArgGlyProLeu                              151015                                                                        GGCAACCAGGGCCGAGGCAACCCGGTGCGGTCGCCCTTGGGTTTTGGG96                            GlyAsnGlnGlyArgGlyAsnProValArgSerProLeuGlyPheGly                              202530                                                                        TCCTACGCCATGACCAGGATCCGAGATACCCTACATCTGGTGGAGTGT144                           SerTyrAlaMetThrArgIleArgAspThrLeuHisLeuValGluCys                              354045                                                                        CCCACACCAGCCATCGAGCCTCCCACCGGGACGTCTGGGTTCTTCCCC192                           ProThrProAlaIleGluProProThrGlyThrSerGlyPhePhePro                              505560                                                                        GGGACGCCGCCTCTCAACAGCTGCATG219                                                GlyThrProProLeuAsnSerCysMet                                                   6570                                                                          (2) INFORMATION FOR SEQ ID NO:144:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:                                     ValProPheValAsnArgThrThrLeuPheThrIleArgGlyProLeu                              151015                                                                        GlyAsnGlnGlyArgGlyAsnProValArgSerProLeuGlyPheGly                              202530                                                                        SerTyrAlaMetThrArgIleArgAspThrLeuHisLeuValGluCys                              354045                                                                        ProThrProAlaIleGluProProThrGlyThrSerGlyPhePhePro                              505560                                                                        GlyThrProProLeuAsnSerCysMet                                                   6570                                                                          (2) INFORMATION FOR SEQ ID NO:145:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 264 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Antigen Clone Y12-10-3                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..264                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:                                     CCCCTCGAGCGGATGCGAACCGGAAGGCACCTCGTGTTCTGCCATTCT48                            ProLeuGluArgMetArgThrGlyArgHisLeuValPheCysHisSer                              151015                                                                        AAGGCTGAGTGCGAGCGCCTTGCTGGCCAGTTCTCCGCTAGGGGGGTC96                            LysAlaGluCysGluArgLeuAlaGlyGlnPheSerAlaArgGlyVal                              202530                                                                        AATGCCATTGCCTATTATAGGGGTAAAGACAGCTCTATCATCAAGGAT144                           AsnAlaIleAlaTyrTyrArgGlyLysAspSerSerIleIleLysAsp                              354045                                                                        GGGGACCTGGTGGTCTGTGCTACAGACGCGCTTTCCACTGGGTACACT192                           GlyAspLeuValValCysAlaThrAspAlaLeuSerThrGlyTyrThr                              505560                                                                        GGAAATTTCGACTCCGTCACCGACTGTGGATTAGTGGTGGAGGAGGTC240                           GlyAsnPheAspSerValThrAspCysGlyLeuValValGluGluVal                              65707580                                                                      GTTGAGGTGACCCTTGATCCCACC264                                                   ValGluValThrLeuAspProThr                                                      85                                                                            (2) INFORMATION FOR SEQ ID NO:146:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 88 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:                                     ProLeuGluArgMetArgThrGlyArgHisLeuValPheCysHisSer                              151015                                                                        LysAlaGluCysGluArgLeuAlaGlyGlnPheSerAlaArgGlyVal                              202530                                                                        AsnAlaIleAlaTyrTyrArgGlyLysAspSerSerIleIleLysAsp                              354045                                                                        GlyAspLeuValValCysAlaThrAspAlaLeuSerThrGlyTyrThr                              505560                                                                        GlyAsnPheAspSerValThrAspCysGlyLeuValValGluGluVal                              65707580                                                                      ValGluValThrLeuAspProThr                                                      85                                                                            (2) INFORMATION FOR SEQ ID NO:147:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 205 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Antigen Clone Y12-15-1                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..205                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:                                     GCTAGATCTGGCAACATGGGGCACAAGGTCTTAATCTTGAACCCCTCA48                            AlaArgSerGlyAsnMetGlyHisLysValLeuIleLeuAsnProSer                              151015                                                                        GTGGCCACTGTGCGGGCCATGGGCCCGTACATGGAGCGGCTGGCGGGT96                            ValAlaThrValArgAlaMetGlyProTyrMetGluArgLeuAlaGly                              202530                                                                        AAACATCCAAGTATATACTGTGGGCATGATACAACTGCTTTCACAAGG144                           LysHisProSerIleTyrCysGlyHisAspThrThrAlaPheThrArg                              354045                                                                        ATCACTGACTCCCCCCTGACGTATTCAACCTATGGGAGGTTTTTGGCC192                           IleThrAspSerProLeuThrTyrSerThrTyrGlyArgPheLeuAla                              505560                                                                        AACCCTAGGCAGA205                                                              AsnProArgGln                                                                  65                                                                            (2) INFORMATION FOR SEQ ID NO:148:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:                                     AlaArgSerGlyAsnMetGlyHisLysValLeuIleLeuAsnProSer                              151015                                                                        ValAlaThrValArgAlaMetGlyProTyrMetGluArgLeuAlaGly                              202530                                                                        LysHisProSerIleTyrCysGlyHisAspThrThrAlaPheThrArg                              354045                                                                        IleThrAspSerProLeuThrTyrSerThrTyrGlyArgPheLeuAla                              505560                                                                        AsnProArgGln                                                                  65                                                                            (2) INFORMATION FOR SEQ ID NO:149:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE4F                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:                                     GCCGCCATGGCTCTCCAAGCGATCGAGAATGC32                                            (2) INFORMATION FOR SEQ ID NO:150:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE4R                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:                                     GCGCGGATCCCAACCCCAATGAGAAAAAGCG31                                             (2) INFORMATION FOR SEQ ID NO:151:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470EXP3F                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:                                     CCGCCATGGGACGCGGACGCTCG23                                                     (2) INFORMATION FOR SEQ ID NO:152:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470EXP3R                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:                                     CGCGGATCCTTACTGTCTTATTGCTTCC28                                                (2) INFORMATION FOR SEQ ID NO:153:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer FV94- 2888F                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:                                     GCGGAATTCTTGGCTCGGGTGGTTGAGTGCTGTG34                                          (2) INFORMATION FOR SEQ ID NO:154:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer FV94- 3216R                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:                                     GCGAAGCTTCCGTCGGATGACAACAGGCGCGG32                                            (2) INFORMATION FOR SEQ ID NO:155:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer FV94- 6521F                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:                                     GCGGAATTCACCTCCGCCTATAAGCTGCTGCGCCAG36                                        (2) INFORMATION FOR SEQ ID NO:156:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer FV94- 7483R                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:                                     GCTGCGGCCGCCCTCCGTCCCACATTGTCTGGATTGGTAACA42                                  (2) INFORMATION FOR SEQ ID NO:157:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer T7F                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:                                     ATTAATACGACTCACTATAGGG22                                                      (2) INFORMATION FOR SEQ ID NO:158:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer T7R                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:                                     CAAGGGGTTATGCTAGTTATTG22                                                      (2) INFORMATION FOR SEQ ID NO:159:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Antigen Clone GE4-8                                   (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..402                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:                                     GCTCTCCAAGCGATCGAGAATGCTGCGAGGATTCTTGAACCGCACATT48                            AlaLeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIle                              151015                                                                        GATGTCATCATGGAGGACTGCAGTACACCCTCTCTTTGTGGTAGTAGC96                            AspValIleMetGluAspCysSerThrProSerLeuCysGlySerSer                              202530                                                                        CGAGAGATGCCTGTATGGGGAGAAGACATCCCCCGTACTCCATCGCCA144                           ArgGluMetProValTrpGlyGluAspIleProArgThrProSerPro                              354045                                                                        GCACTTATCTCGGTTACTGAGAGCAGCTCAGATGAGAAGACCCCGTCG192                           AlaLeuIleSerValThrGluSerSerSerAspGluLysThrProSer                              505560                                                                        GTGTCCTCCTCGCAGGAGGATACCCCGTCCTCTGACTCATTCGAGGTC240                           ValSerSerSerGlnGluAspThrProSerSerAspSerPheGluVal                              65707580                                                                      ATCCAAGAGTCCGAGACAGCCGAAGGGGAGGAAAGTGTCTTCAACGTG288                           IleGlnGluSerGluThrAlaGluGlyGluGluSerValPheAsnVal                              859095                                                                        GCTCTTTCCGTATTAAAAGCCTTATTTCCACAGAGCGACGCGACCAGG336                           AlaLeuSerValLeuLysAlaLeuPheProGlnSerAspAlaThrArg                              100105110                                                                     AAGCTTACCGTCAAGATGTCGTGCTGCGTTGAAAAGAGCGTCACGCGC384                           LysLeuThrValLysMetSerCysCysValGluLysSerValThrArg                              115120125                                                                     TTTTTCTCATTGGGGTTG402                                                         PhePheSerLeuGlyLeu                                                            130                                                                           (2) INFORMATION FOR SEQ ID NO:160:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:                                     AlaLeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIle                              151015                                                                        AspValIleMetGluAspCysSerThrProSerLeuCysGlySerSer                              202530                                                                        ArgGluMetProValTrpGlyGluAspIleProArgThrProSerPro                              354045                                                                        AlaLeuIleSerValThrGluSerSerSerAspGluLysThrProSer                              505560                                                                        ValSerSerSerGlnGluAspThrProSerSerAspSerPheGluVal                              65707580                                                                      IleGlnGluSerGluThrAlaGluGlyGluGluSerValPheAsnVal                              859095                                                                        AlaLeuSerValLeuLysAlaLeuPheProGlnSerAspAlaThrArg                              100105110                                                                     LysLeuThrValLysMetSerCysCysValGluLysSerValThrArg                              115120125                                                                     PhePheSerLeuGlyLeu                                                            130                                                                           (2) INFORMATION FOR SEQ ID NO:161:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1011 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Antigen Clone EXP3-7                                  (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1011                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:                                     ATGGTCTATGGCCCTGGGCAAAGTGTTACCATTGACGGGGAGCGCTAC48                            MetValTyrGlyProGlyGlnSerValThrIleAspGlyGluArgTyr                              151015                                                                        ACCTTGCCTCATCAACTGAGGCTCAGGAATGTGGCACCCTCTGAGGTT96                            ThrLeuProHisGlnLeuArgLeuArgAsnValAlaProSerGluVal                              202530                                                                        TCATCCGAGGTGTCCATTGACATTGGGACGGAGACTGAAGACTCAGAA144                           SerSerGluValSerIleAspIleGlyThrGluThrGluAspSerGlu                              354045                                                                        CTGACTGAGGCCGATCTGCCGCCGGCGGCTGCTGCTCTCCAAGCGATC192                           LeuThrGluAlaAspLeuProProAlaAlaAlaAlaLeuGlnAlaIle                              505560                                                                        GAGAATGCTGCGAGGATTCTTGAACCGCACATTGATGTCATCATGGAG240                           GluAsnAlaAlaArgIleLeuGluProHisIleAspValIleMetGlu                              65707580                                                                      GACTGCAGTACACCCTCTCTTTGTGGTAGTAGCCGAGAGATGCCTGTA288                           AspCysSerThrProSerLeuCysGlySerSerArgGluMetProVal                              859095                                                                        TGGGGAGAAGACATCCCCCGTACTCCATCGCCAGCACTTATCTCGGTT336                           TrpGlyGluAspIleProArgThrProSerProAlaLeuIleSerVal                              100105110                                                                     ACTGAGAGCAGCTCAGATGAGAAGACCCCGTCGGTGTCCTCCTCGCAG384                           ThrGluSerSerSerAspGluLysThrProSerValSerSerSerGln                              115120125                                                                     GAGGATACCCCGTCCTCTGACTCATTCGAGGTCATCCAAGAGTCCGAG432                           GluAspThrProSerSerAspSerPheGluValIleGlnGluSerGlu                              130135140                                                                     ACAGCCGAAGGGGAGGAAAGTGTCTTCAACGTGGCTCTTTCCGTATTA480                           ThrAlaGluGlyGluGluSerValPheAsnValAlaLeuSerValLeu                              145150155160                                                                  AAAGCCTTATTTCCACAGAGCGACGCGACCAGGAAGCTTACCGTCAAG528                           LysAlaLeuPheProGlnSerAspAlaThrArgLysLeuThrValLys                              165170175                                                                     ATGTCGTGCTGCGTTGAAAAGAGCGTCACGCGCTTTTTCTCATTGGGG576                           MetSerCysCysValGluLysSerValThrArgPhePheSerLeuGly                              180185190                                                                     TTGACGGTGGCTGATGTTGCTAGCCTGTGTGAGATGGAAATCCAGAAC624                           LeuThrValAlaAspValAlaSerLeuCysGluMetGluIleGlnAsn                              195200205                                                                     CATACAGCCTATTGTGACCAGGTGCGCACTCCGCTTGAATTGCAGGTT672                           HisThrAlaTyrCysAspGlnValArgThrProLeuGluLeuGlnVal                              210215220                                                                     GGGTGCTTGGTGGGCAATGAACTTACCTTTGAATGTGACAAGTGTGAG720                           GlyCysLeuValGlyAsnGluLeuThrPheGluCysAspLysCysGlu                              225230235240                                                                  GCTAGGCAAGAAACCTTGGCCTCCTTCTCTTACATTTGGTCTGGAGTG768                           AlaArgGlnGluThrLeuAlaSerPheSerTyrIleTrpSerGlyVal                              245250255                                                                     CCGCTGACTAGGGCCACGCCGGCCAAGCCTCCCGTGGTGAGGCCGGTT816                           ProLeuThrArgAlaThrProAlaLysProProValValArgProVal                              260265270                                                                     GGCTCTTTGTTAGTGGCCGACACTACTAAGGTGTATGTTACCAATCCA864                           GlySerLeuLeuValAlaAspThrThrLysValTyrValThrAsnPro                              275280285                                                                     GACAATGTGGGACGGAGGGTGGACAAGGTGACCTTCTGGCGTGCTCCT912                           AspAsnValGlyArgArgValAspLysValThrPheTrpArgAlaPro                              290295300                                                                     AGGGTTCATGATAAGTACCTCGTGGACTCTATTGAGCGCGCTAAGAGG960                           ArgValHisAspLysTyrLeuValAspSerIleGluArgAlaLysArg                              305310315320                                                                  GCCGCTCAAGCCTGCCTAAGCATGGGTTACACTTATGAGGAAGCAATA1008                          AlaAlaGlnAlaCysLeuSerMetGlyTyrThrTyrGluGluAlaIle                              325330335                                                                     AGG1011                                                                       Arg                                                                           (2) INFORMATION FOR SEQ ID NO:162:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 337 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:                                     MetValTyrGlyProGlyGlnSerValThrIleAspGlyGluArgTyr                              151015                                                                        ThrLeuProHisGlnLeuArgLeuArgAsnValAlaProSerGluVal                              202530                                                                        SerSerGluValSerIleAspIleGlyThrGluThrGluAspSerGlu                              354045                                                                        LeuThrGluAlaAspLeuProProAlaAlaAlaAlaLeuGlnAlaIle                              505560                                                                        GluAsnAlaAlaArgIleLeuGluProHisIleAspValIleMetGlu                              65707580                                                                      AspCysSerThrProSerLeuCysGlySerSerArgGluMetProVal                              859095                                                                        TrpGlyGluAspIleProArgThrProSerProAlaLeuIleSerVal                              100105110                                                                     ThrGluSerSerSerAspGluLysThrProSerValSerSerSerGln                              115120125                                                                     GluAspThrProSerSerAspSerPheGluValIleGlnGluSerGlu                              130135140                                                                     ThrAlaGluGlyGluGluSerValPheAsnValAlaLeuSerValLeu                              145150155160                                                                  LysAlaLeuPheProGlnSerAspAlaThrArgLysLeuThrValLys                              165170175                                                                     MetSerCysCysValGluLysSerValThrArgPhePheSerLeuGly                              180185190                                                                     LeuThrValAlaAspValAlaSerLeuCysGluMetGluIleGlnAsn                              195200205                                                                     HisThrAlaTyrCysAspGlnValArgThrProLeuGluLeuGlnVal                              210215220                                                                     GlyCysLeuValGlyAsnGluLeuThrPheGluCysAspLysCysGlu                              225230235240                                                                  AlaArgGlnGluThrLeuAlaSerPheSerTyrIleTrpSerGlyVal                              245250255                                                                     ProLeuThrArgAlaThrProAlaLysProProValValArgProVal                              260265270                                                                     GlySerLeuLeuValAlaAspThrThrLysValTyrValThrAsnPro                              275280285                                                                     AspAsnValGlyArgArgValAspLysValThrPheTrpArgAlaPro                              290295300                                                                     ArgValHisAspLysTyrLeuValAspSerIleGluArgAlaLysArg                              305310315320                                                                  AlaAlaGlnAlaCysLeuSerMetGlyTyrThrTyrGluGluAlaIle                              325330335                                                                     Arg                                                                           (2) INFORMATION FOR SEQ ID NO:163:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 351 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Antigen Clone GENS2b-1                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..351                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:                                     TTGGCTCGGGTGGTTGAGTGCTGTGTGATGGCGGGTGAGAAGGCCACA48                            LeuAlaArgValValGluCysCysValMetAlaGlyGluLysAlaThr                              151015                                                                        ACCGTCCGGCTGGTCTCCAAGATGTGTGCGAGAGGAGCTTATTTGTTC96                            ThrValArgLeuValSerLysMetCysAlaArgGlyAlaTyrLeuPhe                              202530                                                                        GATCATATGGGCTCTTTTTCGCGTGCTGTCAAGGAGCGCCTGTTGGAA144                           AspHisMetGlySerPheSerArgAlaValLysGluArgLeuLeuGlu                              354045                                                                        TGGGACGCAGCTCTTGAACCTCTGTCATTCACTAGGACGGACTGTCGC192                           TrpAspAlaAlaLeuGluProLeuSerPheThrArgThrAspCysArg                              505560                                                                        ATCATACGGGATGCCGCGAGGACTTTGTCCTGCGGGCAGTGCGTCATG240                           IleIleArgAspAlaAlaArgThrLeuSerCysGlyGlnCysValMet                              65707580                                                                      GGTTTACCCGTGGTTGCGCGCCGTGGTGATGAGGTTCTCATCGGCGTC288                           GlyLeuProValValAlaArgArgGlyAspGluValLeuIleGlyVal                              859095                                                                        TTCCAGGATGTGAATCATTTGCCTCCCGGGTTTGTTCCGACCGCGCCT336                           PheGlnAspValAsnHisLeuProProGlyPheValProThrAlaPro                              100105110                                                                     GTTGTCATCCGACGG351                                                            ValValIleArgArg                                                               115                                                                           (2) INFORMATION FOR SEQ ID NO:164:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:                                     LeuAlaArgValValGluCysCysValMetAlaGlyGluLysAlaThr                              151015                                                                        ThrValArgLeuValSerLysMetCysAlaArgGlyAlaTyrLeuPhe                              202530                                                                        AspHisMetGlySerPheSerArgAlaValLysGluArgLeuLeuGlu                              354045                                                                        TrpAspAlaAlaLeuGluProLeuSerPheThrArgThrAspCysArg                              505560                                                                        IleIleArgAspAlaAlaArgThrLeuSerCysGlyGlnCysValMet                              65707580                                                                      GlyLeuProValValAlaArgArgGlyAspGluValLeuIleGlyVal                              859095                                                                        PheGlnAspValAsnHisLeuProProGlyPheValProThrAlaPro                              100105110                                                                     ValValIleArgArg                                                               115                                                                           (2) INFORMATION FOR SEQ ID NO:165:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 993 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Antigen Clone GENS5a-3                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..993                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:                                     ACCTCCGCCTATAAGCTGCTGCGCCAGCAAATCCTATCGGCTGCTGTA48                            ThrSerAlaTyrLysLeuLeuArgGlnGlnIleLeuSerAlaAlaVal                              151015                                                                        GCTGAGCCCTACTACGTCGACGGCATTCCGGTCTCATGGGACGCGGAC96                            AlaGluProTyrTyrValAspGlyIleProValSerTrpAspAlaAsp                              202530                                                                        GCTCGTGCGCCCGCCATGGTCTATGGCCCTGGGCAAAGTGTTACCATT144                           AlaArgAlaProAlaMetValTyrGlyProGlyGlnSerValThrIle                              354045                                                                        GACGGGGAGCGCTACACCTTGCCTCATCAACTGAGGCTCAGGAATGTG192                           AspGlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnVal                              505560                                                                        GCACCCTCTGAGGTTTCATCCGAGGTGTCCATTGACATTGGGACGGAG240                           AlaProSerGluValSerSerGluValSerIleAspIleGlyThrGlu                              65707580                                                                      ACTGAAGACTCAGAACTGACTGAGGCCGATCTGCCGCCGGCGGCTGCT288                           ThrGluAspSerGluLeuThrGluAlaAspLeuProProAlaAlaAla                              859095                                                                        GCTCTCCAAGCGATCGAGAATGCTGCGAGGATTCTTGAACCGCACATT336                           AlaLeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIle                              100105110                                                                     GATGTCATCATGGAGGACTGCAGTACACCCTCTCTTTGTGGTAGTAGC384                           AspValIleMetGluAspCysSerThrProSerLeuCysGlySerSer                              115120125                                                                     CGAGAGATGCCTGTATGGGGAGAAGACATCCCCCGTACTCCATCGCCA432                           ArgGluMetProValTrpGlyGluAspIleProArgThrProSerPro                              130135140                                                                     GCACTTATCTCGGTTACTGAGAGCAGCTCAGATGAGAAGACCCCGTCG480                           AlaLeuIleSerValThrGluSerSerSerAspGluLysThrProSer                              145150155160                                                                  GTGTCCTCCTCGCAGGAGGATACCCCGTCCTCTGACTCATTCGAGGTC528                           ValSerSerSerGlnGluAspThrProSerSerAspSerPheGluVal                              165170175                                                                     ATCCAAGAGTCCGAGACAGCCGAAGGGGAGGAAAGTGTCTTCAACGTG576                           IleGlnGluSerGluThrAlaGluGlyGluGluSerValPheAsnVal                              180185190                                                                     GCTCTTTCCGTATTAAAAGCCTTATTTCCACAGAGCGACGCGACCAGG624                           AlaLeuSerValLeuLysAlaLeuPheProGlnSerAspAlaThrArg                              195200205                                                                     AAGCTTACCGTCAAGATGTCGTGCTGCGTTGAAAAGAGCGTCACGCGC672                           LysLeuThrValLysMetSerCysCysValGluLysSerValThrArg                              210215220                                                                     TTTTTCTCATTGGGGTTGACGGTGGCTGATGTTGCTAGCCTGTGTGAG720                           PhePheSerLeuGlyLeuThrValAlaAspValAlaSerLeuCysGlu                              225230235240                                                                  ATGGAAATCCAGAACCATACAGCCTATTGTGACCAGGTGCGCACTCCG768                           MetGluIleGlnAsnHisThrAlaTyrCysAspGlnValArgThrPro                              245250255                                                                     CTTGAATTGCAGGTTGGGTGCTTGGTGGGCAATGAACTTACCTTTGAA816                           LeuGluLeuGlnValGlyCysLeuValGlyAsnGluLeuThrPheGlu                              260265270                                                                     TGTGACAAGTGTGAGGCTAGGCAAGAAACCTTGGCCTCCTTCTCTTAC864                           CysAspLysCysGluAlaArgGlnGluThrLeuAlaSerPheSerTyr                              275280285                                                                     ATTTGGTCTGGAGTGCCGCTGACTAGGGCCACGCCGGCCAAGCCTCCC912                           IleTrpSerGlyValProLeuThrArgAlaThrProAlaLysProPro                              290295300                                                                     GTGGTGAGGCCGGTTGGCTCTTTGTTAGTGGCCGACACTACTAAGGTG960                           ValValArgProValGlySerLeuLeuValAlaAspThrThrLysVal                              305310315320                                                                  TATGTTACCAATCCAGACAATGTGGGACGGAGG993                                          TyrValThrAsnProAspAsnValGlyArgArg                                             325330                                                                        (2) INFORMATION FOR SEQ ID NO:166:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 331 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:                                     ThrSerAlaTyrLysLeuLeuArgGlnGlnIleLeuSerAlaAlaVal                              151015                                                                        AlaGluProTyrTyrValAspGlyIleProValSerTrpAspAlaAsp                              202530                                                                        AlaArgAlaProAlaMetValTyrGlyProGlyGlnSerValThrIle                              354045                                                                        AspGlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnVal                              505560                                                                        AlaProSerGluValSerSerGluValSerIleAspIleGlyThrGlu                              65707580                                                                      ThrGluAspSerGluLeuThrGluAlaAspLeuProProAlaAlaAla                              859095                                                                        AlaLeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIle                              100105110                                                                     AspValIleMetGluAspCysSerThrProSerLeuCysGlySerSer                              115120125                                                                     ArgGluMetProValTrpGlyGluAspIleProArgThrProSerPro                              130135140                                                                     AlaLeuIleSerValThrGluSerSerSerAspGluLysThrProSer                              145150155160                                                                  ValSerSerSerGlnGluAspThrProSerSerAspSerPheGluVal                              165170175                                                                     IleGlnGluSerGluThrAlaGluGlyGluGluSerValPheAsnVal                              180185190                                                                     AlaLeuSerValLeuLysAlaLeuPheProGlnSerAspAlaThrArg                              195200205                                                                     LysLeuThrValLysMetSerCysCysValGluLysSerValThrArg                              210215220                                                                     PhePheSerLeuGlyLeuThrValAlaAspValAlaSerLeuCysGlu                              225230235240                                                                  MetGluIleGlnAsnHisThrAlaTyrCysAspGlnValArgThrPro                              245250255                                                                     LeuGluLeuGlnValGlyCysLeuValGlyAsnGluLeuThrPheGlu                              260265270                                                                     CysAspLysCysGluAlaArgGlnGluThrLeuAlaSerPheSerTyr                              275280285                                                                     IleTrpSerGlyValProLeuThrArgAlaThrProAlaLysProPro                              290295300                                                                     ValValArgProValGlySerLeuLeuValAlaAspThrThrLysVal                              305310315320                                                                  TyrValThrAsnProAspAsnValGlyArgArg                                             325330                                                                        (2) INFORMATION FOR SEQ ID NO:167:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 536 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 3'-end                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:                                     CTGAGCGACCTCAAGCTCCCTGGCTTAGCAGTCCACCGAAAGAAGGCCGGGGCGTTGCGA60                ACACGCATGCTCCGCTCGCGCGGTTGGGCTGAGTTGGCTAGGGGCTTGTTGTGGCATCCA120               GGCCTACGGCTTCCTCCCCCTGAGATTGCTGGTATCCCGGGGGGTTTCCCTCTCTCCCCC180               CCCTATATGGGGGTGGTACATCAATTGGATTTCACAAGCCAGAGGAGTCGCTGGCGGTGG240               TTGGGGTTCTTAGCCCTGCTCATCGTAGCCCTCTTCGGGTGAACTAAATTCATCTGTTGC300               GGCAAGGTCTGGTGACTGATCATCACCGGAGGAGGTTCCCGCCCTCCCCGCCCCAGGGGT360               CTCCCCGCTGGGTAAAAAGGGCCCGGCCTTGGGAGGCATGGTGGTTACTAACCCCCTGGC420               AGGGTCAAAGCCTGATGGTGCTAATGCACTGCCACTTCGGTGGCGGGTCGCTACCTTATA480               GCGTAATCCGTGACTACGGGCTGCTCGCAGAGCCCTCCCCGGATGGGGCACAGTGC536                   (2) INFORMATION FOR SEQ ID NO:168:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 594 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Individual Clone MP3-3                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:                                     CTGAGCGACCTCAAGCTCCCTGGCTTAGCAGTCCACCGAAAGAAGGCCGGGGCGTTGCGA60                ACACGCATGCTCCGCTCGCGCGGTTGGGCTGAGTTGGCTAGGGGCTTGTTGTGGCATCCA120               GGCCTACGGCTTCCTCCCCCTGAGATTGCTGGTATCCCGGGGGGTTTCCCTCTCTCCCCC180               CCCTATATGGGGGTGGTACACCAATTGGATTTCACAAGCCAGAGGAGTCGCTGGCGGTGG240               TTGGGGTTCTTAGCCCTGCTCATCGTAGCCCTCTTCGGGTGAACTAAATTCATCTGTTGC300               GGCAAGGTCTGGTGACTGATCATCACCGGAGGAGGTTCCCGCCCTCCCCGCCCCAGGGGT360               CTCCCCGCTGGGTAAAAAGGGCCCGGCCTTGGGAGGCATGGTGGTTACTAACCCCCTGGC420               AGGGTCAAAGCCTGATGGTGCTAATGCACTGCCACTTCGGTGGCGGGTCGCTACCTTATA480               GCGTAATCCGTGACTACGGGCTGCTCGCAGAGCCCTCCCCGGATGGGGCACAGTGCACTG540               TGATCTGAAGGGGTGCACCCCGGGAAGAGCTCGGCCCGAAGGCCGGCTTCTACT594                     (2) INFORMATION FOR SEQ ID NO:169:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 594 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Individual Clone MP3-7                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:                                     CTGAGCGACCTCAAGCTCCCTGGCTTAGCAGTCCACCGAAAGAAGGCCGGGGCGTTGCGA60                ACACGCATGCTCCGCTCGCGCGGTTGGGCTGAGTTGGCTAGGGGCTTGTTGTGGCATCCA120               GGCCTACGGCTTCCTCCCCCTGAGATTGCTGGTGTCCCGGGGGGTTTCCCTCTCTCCCCC180               CCCTATATGGGGGTGGTACACCAATTGGATTTCACAAGCCAGAGGAGTCGCTGGCGGTGG240               TTGGGGTTCTTAGCCCTGCTCATCGTAGCCCTCTTCGGGTGAACTAAATTCATCTGTTGC300               GGCAAGGTCTGGTGACTGATCATCACCGGAGGAGGTTCCCGCCCTCCCCGCCCCAGGGGT360               CTCCCCGCTGGGTAAAAAGGGCCCGGCCTTGGGAGGCATGGTGGTTACTAACCCCCTGGC420               AGGGTCAAAGCCTGATGGTGCTAATGCACTGCCACTTCGGTGGCGGGTCGCTACCTTATA480               GCGTAATCCGTGACTACGGGCTGCTCGCAGAGCCCTCCCCGGATGGGGCACAGTGCACTG540               TGATCTGAAGGGGTGCACCCCGGTAAGAGCTCGGCCCGAAGGCCGGGTTCTACT594                     (2) INFORMATION FOR SEQ ID NO:170:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GV5446IRT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:                                     CGGTCCCTCGAACTCCAGCGAGTCTTTTTTTTTTTTTTT39                                     (2) INFORMATION FOR SEQ ID NO:171:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GV59- 5446F                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:                                     CTGAGCGACCTCAAGCTCCCTGGC24                                                    (2) INFORMATION FOR SEQ ID NO:172:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GV- 5446IR                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:                                     CGGTCCCTCGAACTCCAGCGAGTC24                                                    (2) INFORMATION FOR SEQ ID NO:173:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Probe E5-7- PRB                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:                                     CGTAGCCCTCGGGTGAACTAAAT23                                                     (2) INFORMATION FOR SEQ ID NO:174:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Race Anchor Sequence                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:                                     CACGAATTCACTATCGATTCTGGAACCTTCAGAGG35                                         (2) INFORMATION FOR SEQ ID NO:175:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 736 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Consensus Sequence 5'-end                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:                                     ACGTGGGGGAGTTGATCCCCCCCCCCCGGCACTGGGTGCAAGCCCCAGAAACCGACGCCT60                ATCTAAGTAGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGG120               GTGATGACAGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGT180               CTTAAGAGAAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGT240               GTTGGCCCTACCGGTGGGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGT300               TACCCACCTGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTG360               ACCAATAGGCGTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGAGAGGG420               ACTCCAAGTCCCGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACCCAGCTCCGCGG480               CGGCCTGCAGCCGGGGTAGCCCAAGAATCCTTCGGGTGAGGGCGGGTGGCATTTCCTTTT540               TCTATACCATCATGGCAGTCCTTCTGCTCCTTCTCGTGGTTGAGGCCGGGGCCATTCTGG600               CCCCGGCCACCCACGCTTGTCGAGCGAATGGGCAATATTTCCTCACAAATTGTTGTGCCC660               CGGAGGACATCGGGTTCTGCCTGGAGGGTGGATGCCTGGTGGCCCTGGGGTGCACGATTT720               GCACTGACCAATGCTG736                                                           (2) INFORMATION FOR SEQ ID NO:176:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 688 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV Variant BG34                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 272..688                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:                                     GACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGACAGGGTTGGT60                AGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAGAAGGTTAAG120               ATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCCTACCGGTGT180               GAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACCTGGGCAAAC240               GACGCCCACGTACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAGG292                       MetProLeuLeuAlaAsnArg                                                         15                                                                            AGTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGAGTCACGGGG340                           SerIleArgArgValAspLysAspGlnTrpGlyProGlyValThrGly                              101520                                                                        ATGGACCCCGGGCTCTGCCCTTCCCGGTGGAACGGGAAACGCATGGGG388                           MetAspProGlyLeuCysProSerArgTrpAsnGlyLysArgMetGly                              253035                                                                        CCACCCAGCTCCGCGGCGGCCTGCAGCCGGGGTAGCCCAAGAACCCTT436                           ProProSerSerAlaAlaAlaCysSerArgGlySerProArgThrLeu                              40455055                                                                      CGGGTGAGGGCGGGTGGCATTTCTCTTTTCTGTATCATCATGGCAGTC484                           ArgValArgAlaGlyGlyIleSerLeuPheCysIleIleMetAlaVal                              606570                                                                        CTCCTGCTCCTTCTCGTGGTTGAGGCCGGGGCCATTCTGGCCCCGGCC532                           LeuLeuLeuLeuLeuValValGluAlaGlyAlaIleLeuAlaProAla                              758085                                                                        ACCCACGCTTGTCGAGCGAATGGACAATATTTCCTCACAAACTGTTGC580                           ThrHisAlaCysArgAlaAsnGlyGlnTyrPheLeuThrAsnCysCys                              9095100                                                                       GCCCTCGAGGACATCGGGTTCTGCCTGGAAGGCGGGTGCCTGGTGGCC628                           AlaLeuGluAspIleGlyPheCysLeuGluGlyGlyCysLeuValAla                              105110115                                                                     TTAGGGTGCACCATTTGCACTGACCGTTGCTGGCCACTGTATCAGGCG676                           LeuGlyCysThrIleCysThrAspArgCysTrpProLeuTyrGlnAla                              120125130135                                                                  GGTTTGGCTGTG688                                                               GlyLeuAlaVal                                                                  (2) INFORMATION FOR SEQ ID NO:177:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 139 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:                                     MetProLeuLeuAlaAsnArgSerIleArgArgValAspLysAspGln                              151015                                                                        TrpGlyProGlyValThrGlyMetAspProGlyLeuCysProSerArg                              202530                                                                        TrpAsnGlyLysArgMetGlyProProSerSerAlaAlaAlaCysSer                              354045                                                                        ArgGlySerProArgThrLeuArgValArgAlaGlyGlyIleSerLeu                              505560                                                                        PheCysIleIleMetAlaValLeuLeuLeuLeuLeuValValGluAla                              65707580                                                                      GlyAlaIleLeuAlaProAlaThrHisAlaCysArgAlaAsnGlyGln                              859095                                                                        TyrPheLeuThrAsnCysCysAlaLeuGluAspIleGlyPheCysLeu                              100105110                                                                     GluGlyGlyCysLeuValAlaLeuGlyCysThrIleCysThrAspArg                              115120125                                                                     CysTrpProLeuTyrGlnAlaGlyLeuAlaVal                                             130135                                                                        (2) INFORMATION FOR SEQ ID NO:178:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 663 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV Variant T55806                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 271..663                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:                                     GACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCCAGGGTTGGT60                AGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGAGAAGGTTAAG120               ATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCCTACCGGTGG180               AATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACCTGGGCAAACG240               ACGCTCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAGGTTT294                     MetSerLeuLeuThrAsnArgPhe                                                      15                                                                            ATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGTTACGGGGACG342                           IleArgArgValAspLysAspGlnTrpGlyProGlyValThrGlyThr                              101520                                                                        GACCCCGAACCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCA390                           AspProGluProCysProSerArgTrpAlaGlyLysCysMetGlyPro                              25303540                                                                      CCCAGCTCCGCGGCGGCCTGCAGCCGGGGTAGCCCAAGAATCCTTCGG438                           ProSerSerAlaAlaAlaCysSerArgGlySerProArgIleLeuArg                              455055                                                                        GTGAGGGCGGGTGGCATTTCTCTTTTCTATACCATCATGGCAGTCCTT486                           ValArgAlaGlyGlyIleSerLeuPheTyrThrIleMetAlaValLeu                              606570                                                                        CTGCTCTTCTTCGTGGTTGAGGCCGGGGCGATTCTCGCCCCGGCCACC534                           LeuLeuPhePheValValGluAlaGlyAlaIleLeuAlaProAlaThr                              758085                                                                        CACGCTTGTCGGGCGAATGGGCAATATTTCCTCACAAATTGTTGCGCC582                           HisAlaCysArgAlaAsnGlyGlnTyrPheLeuThrAsnCysCysAla                              9095100                                                                       CCAGAGGATGTTGGGTTCTGCCTGGAGGGCGGATGCCTGGTGGCTCTG630                           ProGluAspValGlyPheCysLeuGluGlyGlyCysLeuValAlaLeu                              105110115120                                                                  GGGTGTACGATTTGCACTGACCGTTGCTGGCCA663                                          GlyCysThrIleCysThrAspArgCysTrpPro                                             125130                                                                        (2) INFORMATION FOR SEQ ID NO:179:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 131 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:                                     MetSerLeuLeuThrAsnArgPheIleArgArgValAspLysAspGln                              151015                                                                        TrpGlyProGlyValThrGlyThrAspProGluProCysProSerArg                              202530                                                                        TrpAlaGlyLysCysMetGlyProProSerSerAlaAlaAlaCysSer                              354045                                                                        ArgGlySerProArgIleLeuArgValArgAlaGlyGlyIleSerLeu                              505560                                                                        PheTyrThrIleMetAlaValLeuLeuLeuPhePheValValGluAla                              65707580                                                                      GlyAlaIleLeuAlaProAlaThrHisAlaCysArgAlaAsnGlyGln                              859095                                                                        TyrPheLeuThrAsnCysCysAlaProGluAspValGlyPheCysLeu                              100105110                                                                     GluGlyGlyCysLeuValAlaLeuGlyCysThrIleCysThrAspArg                              115120125                                                                     CysTrpPro                                                                     130                                                                           (2) INFORMATION FOR SEQ ID NO:180:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 632 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV Variant EB20-2                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 271..632                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:180:                                     GACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCCAGGGTTGGT60                AGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGAGAAGGTTAAG120               ATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCCTACCGGTGT180               AATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACCTGGGCAAACG240               ACGCCCACGTACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAGGAGT294                     MetProLeuLeuAlaAsnArgSer                                                      15                                                                            TATCTCCGGCGAGTTGGCAAGGACCAGTGGGGGCCGGGGGTTACGGGG342                           TyrLeuArgArgValGlyLysAspGlnTrpGlyProGlyValThrGly                              101520                                                                        AAGGACCCCGAACCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGG390                           LysAspProGluProCysProSerArgTrpAlaGlyLysCysMetGly                              25303540                                                                      CCACCCAGCTCCGCGGCGGCCTGCAGCCGGGGTAGCCCAAAAAACCTT438                           ProProSerSerAlaAlaAlaCysSerArgGlySerProLysAsnLeu                              455055                                                                        CGGGTGAGGGCGGGTGGCATTTTCTTTTCCTATACCATCATGGCAGTC486                           ArgValArgAlaGlyGlyIlePhePheSerTyrThrIleMetAlaVal                              606570                                                                        CTTCTGCTCCTTCTCGTGGTTGAGGCCGGGGCCATTTTGGCCCCGGCC534                           LeuLeuLeuLeuLeuValValGluAlaGlyAlaIleLeuAlaProAla                              758085                                                                        ACCCACGCTTGCAGAGCTAATGGGCAATATTTCCTCACAAACTGTTGT582                           ThrHisAlaCysArgAlaAsnGlyGlnTyrPheLeuThrAsnCysCys                              9095100                                                                       GCCTTGGAGGACATCGGGTTCTGCCTGGAAGGCGGATGCTTGGTGGCGCT632                         AlaLeuGluAspIleGlyPheCysLeuGluGlyGlyCysLeuValAla                              105110115120                                                                  (2) INFORMATION FOR SEQ ID NO:181:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181:                                     MetProLeuLeuAlaAsnArgSerTyrLeuArgArgValGlyLysAsp                              151015                                                                        GlnTrpGlyProGlyValThrGlyLysAspProGluProCysProSer                              202530                                                                        ArgTrpAlaGlyLysCysMetGlyProProSerSerAlaAlaAlaCys                              354045                                                                        SerArgGlySerProLysAsnLeuArgValArgAlaGlyGlyIlePhe                              505560                                                                        PheSerTyrThrIleMetAlaValLeuLeuLeuLeuLeuValValGlu                              65707580                                                                      AlaGlyAlaIleLeuAlaProAlaThrHisAlaCysArgAlaAsnGly                              859095                                                                        GlnTyrPheLeuThrAsnCysCysAlaLeuGluAspIleGlyPheCys                              100105110                                                                     LeuGluGlyGlyCysLeuValAla                                                      115120                                                                        (2) INFORMATION FOR SEQ ID NO:182:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9103 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-JC Variant                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 276..9005                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:                                     CAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGACAGGGT60                TGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAGAAGGT120               TAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCCTACCG180               GTGGGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTAACCCGCCTGGGC240               AAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCGCTCTTGACCAAT293                      MetSerLeuLeuThrAsn                                                            15                                                                            AGGCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGTTTATG341                           ArgLeuSerArgArgValAspLysAspGlnTrpGlyProGlyPheMet                              101520                                                                        GGGAAGGACCCCAAACCCTGCCCTTCCCGGCGGACCGGGAAATGCATG389                           GlyLysAspProLysProCysProSerArgArgThrGlyLysCysMet                              253035                                                                        GGGCCACCCAGCTCCGCGGCGGCCTGCAGCCGGGGTAGCCCAAGAATC437                           GlyProProSerSerAlaAlaAlaCysSerArgGlySerProArgIle                              404550                                                                        CTTCGGGTGAGGGCGGGTGGCATTTCTCTTCCTTATACCATCATGGAA485                           LeuArgValArgAlaGlyGlyIleSerLeuProTyrThrIleMetGlu                              55606570                                                                      GCCCTCCTGTTCCTCCTCGGGGTGGAGGCCGGGGCCATTCTGGCCCCG533                           AlaLeuLeuPheLeuLeuGlyValGluAlaGlyAlaIleLeuAlaPro                              758085                                                                        GCCACCCACGCTTGTCGAGCGAATGGGCAATATTTCCTCACAAACTGT581                           AlaThrHisAlaCysArgAlaAsnGlyGlnTyrPheLeuThrAsnCys                              9095100                                                                       TGTGCTCCAGAGGACATTGGGTTCTGCCTCGAAGGCGGTTGCCTTGTG629                           CysAlaProGluAspIleGlyPheCysLeuGluGlyGlyCysLeuVal                              105110115                                                                     GCCCTGGGGTGCACAGTTTGCACTGACCGATGCTGGCCGCTGTATCAG677                           AlaLeuGlyCysThrValCysThrAspArgCysTrpProLeuTyrGln                              120125130                                                                     GCGGGCTTGGCTGTGCGGCCTGGCAAGTCCGCAGCCCAGCTGGTGGGG725                           AlaGlyLeuAlaValArgProGlyLysSerAlaAlaGlnLeuValGly                              135140145150                                                                  CAACTGGGTGGCCTCTACGGGCCCTTGTCGGTGTCGGCCTACGTGGCC773                           GlnLeuGlyGlyLeuTyrGlyProLeuSerValSerAlaTyrValAla                              155160165                                                                     GGCATCCTGGGCCTGGGTGAGGTGTACTCGGGTGTCCTAACAGTTGGT821                           GlyIleLeuGlyLeuGlyGluValTyrSerGlyValLeuThrValGly                              170175180                                                                     GTTGCGTTGACGCGCCGGGTCTACCCGATGCCCAACCTGACGTGTGCA869                           ValAlaLeuThrArgArgValTyrProMetProAsnLeuThrCysAla                              185190195                                                                     GTAGAGTGTGAGCTTAAGTGGGAAAGTGAGTTTTGGAGATGGACTGAG917                           ValGluCysGluLeuLysTrpGluSerGluPheTrpArgTrpThrGlu                              200205210                                                                     CAGCTGGCCTCCAATTACTGGATTCTGGAATACCTTTGGAAGGTCCCG965                           GlnLeuAlaSerAsnTyrTrpIleLeuGluTyrLeuTrpLysValPro                              215220225230                                                                  TTTGACTTCTGGAGAGGCGTGCTAAGCCTGACTCCCTTGCTGGTTTGC1013                          PheAspPheTrpArgGlyValLeuSerLeuThrProLeuLeuValCys                              235240245                                                                     GTGGCCGCGTTGCTGCTGCTGGAGCAACGGATTGTCATGGTCTTCCTG1061                          ValAlaAlaLeuLeuLeuLeuGluGlnArgIleValMetValPheLeu                              250255260                                                                     TTGGTGACGATGGCCGGGATGTCGCAAGGCGCTCCGGCCTCCGTTTTG1109                          LeuValThrMetAlaGlyMetSerGlnGlyAlaProAlaSerValLeu                              265270275                                                                     GGGTCTCGCCCCTTTGACTACGGGTTGACATGGCAGTCTTGTTCCTGC1157                          GlySerArgProPheAspTyrGlyLeuThrTrpGlnSerCysSerCys                              280285290                                                                     AGGGCTAATGGGTCGCGCTATACTACTGGGGAGAAGGTGTGGGACCGT1205                          ArgAlaAsnGlySerArgTyrThrThrGlyGluLysValTrpAspArg                              295300305310                                                                  GGGAACGTCACGCTCCTGTGTGACTGCCCCAACGGCCCCTGGGTGTGG1253                          GlyAsnValThrLeuLeuCysAspCysProAsnGlyProTrpValTrp                              315320325                                                                     TTGCCGGCCTTTTGCCAAGCAATCGGCTGGGGCGATCCCATCACTCAT1301                          LeuProAlaPheCysGlnAlaIleGlyTrpGlyAspProIleThrHis                              330335340                                                                     TGGAGCCACGGCCAAAATCGGTGGCCCCTCTCATGCCCCCAGTATGTC1349                          TrpSerHisGlyGlnAsnArgTrpProLeuSerCysProGlnTyrVal                              345350355                                                                     TATGGGTCTGTTTCAGTCACTTGCGTGTGGGGTTCCGTCTCTTGGTTT1397                          TyrGlySerValSerValThrCysValTrpGlySerValSerTrpPhe                              360365370                                                                     GCCTCGACTGGCGGTCGCGACTCGAAGATCGATGTGTGGAGTCTGGTG1445                          AlaSerThrGlyGlyArgAspSerLysIleAspValTrpSerLeuVal                              375380385390                                                                  CCGGTTGGTTCCGCCAGCTGCACCATAGCCGCTCTTGGATCGTCGGAT1493                          ProValGlySerAlaSerCysThrIleAlaAlaLeuGlySerSerAsp                              395400405                                                                     CGGGACACGGTAGTTGAGCTCTCCGAGTGGGGAGTCCCGTGCGCAACG1541                          ArgAspThrValValGluLeuSerGluTrpGlyValProCysAlaThr                              410415420                                                                     TGCATTCTGGATCGTCGGCCGGCCTCGTGCGGCACCTGTGTGAGAGAC1589                          CysIleLeuAspArgArgProAlaSerCysGlyThrCysValArgAsp                              425430435                                                                     TGCTGGCCCGAAACCGGGTCGGTTAGGTTTCCATTCCATCGGTGCGGC1637                          CysTrpProGluThrGlySerValArgPheProPheHisArgCysGly                              440445450                                                                     GCGGGGCCTAAGCTGACAAAGGACTTGGAAGCTGTGCCCTTCGTCAAT1685                          AlaGlyProLysLeuThrLysAspLeuGluAlaValProPheValAsn                              455460465470                                                                  AGGACAACTCCCTTCACCATAAGGGGCCCCCTGGGCAACCAGGGGAGA1733                          ArgThrThrProPheThrIleArgGlyProLeuGlyAsnGlnGlyArg                              475480485                                                                     GGCAACCCGGTGCGGTCGCCCTTGGGTTTTGGGTCCTACGCCATGACC1781                          GlyAsnProValArgSerProLeuGlyPheGlySerTyrAlaMetThr                              490495500                                                                     AAGATCCGAGACTCCTTACATTTGGTGAAATGTCCCACACCAGCCATT1829                          LysIleArgAspSerLeuHisLeuValLysCysProThrProAlaIle                              505510515                                                                     GAGCCTCCCACCGGGACGTTTGGGTTCTTCCCCGGAGTGCCGCCTCTT1877                          GluProProThrGlyThrPheGlyPhePheProGlyValProProLeu                              520525530                                                                     AACAACTGCCTGCTGTTGGGCACGGAAGTGTCCGAAGCGCTGGGCGGG1925                          AsnAsnCysLeuLeuLeuGlyThrGluValSerGluAlaLeuGlyGly                              535540545550                                                                  GCCGGCCTCACGGGGGGGTTCTATGAACCCCTGGTGCGCAGGCGTTCG1973                          AlaGlyLeuThrGlyGlyPheTyrGluProLeuValArgArgArgSer                              555560565                                                                     GAGCTGATGGGGCGCCGAAATCCGGTTTGCCCGGGGTTTGCATGGCTG2021                          GluLeuMetGlyArgArgAsnProValCysProGlyPheAlaTrpLeu                              570575580                                                                     TCCTCGGGTCGACCTGACGGGTTTATACACGTCCAGGGCCACTTGCAG2069                          SerSerGlyArgProAspGlyPheIleHisValGlnGlyHisLeuGln                              585590595                                                                     GAGGTCGATGCTGGCAACTTCATCCCTCCACCTCGCTGGTTGCTCTTG2117                          GluValAspAlaGlyAsnPheIleProProProArgTrpLeuLeuLeu                              600605610                                                                     GACTTTGTGTTTGTCCTGTTATACCTGATGAAGCTGGCTGAGGCACGG2165                          AspPheValPheValLeuLeuTyrLeuMetLysLeuAlaGluAlaArg                              615620625630                                                                  CTGGTCCCGTTGATCTTGCTTCTGCTGTGGTGGTGGGTGAACCAGTTG2213                          LeuValProLeuIleLeuLeuLeuLeuTrpTrpTrpValAsnGlnLeu                              635640645                                                                     GCAGTCCTTGGACTGCCGGCTGTGGACGCCGCCGTGGCTGGTGAGGTC2261                          AlaValLeuGlyLeuProAlaValAspAlaAlaValAlaGlyGluVal                              650655660                                                                     TTCGCGGGCCCGGCCCTGTCGTGGTGTCTGGGCCTCCCCACCGTTAGT2309                          PheAlaGlyProAlaLeuSerTrpCysLeuGlyLeuProThrValSer                              665670675                                                                     ATGATCCTGGGCTTAGCAAACCTGGTGTTGTATTTCCGGTGGATGGGT2357                          MetIleLeuGlyLeuAlaAsnLeuValLeuTyrPheArgTrpMetGly                              680685690                                                                     CCCCAACGCCTCATGTTCCTCGTGTTGTGGAAGCTCGCTCGGGGAGCC2405                          ProGlnArgLeuMetPheLeuValLeuTrpLysLeuAlaArgGlyAla                              695700705710                                                                  TTCCCGCTGGCACTTCTGATGGGGATCTCGGCAACCCGCGGGCGCACC2453                          PheProLeuAlaLeuLeuMetGlyIleSerAlaThrArgGlyArgThr                              715720725                                                                     TCGGTGCTCGGGGCCGAGTTCTGCTTCGATGTCACATTCGAGGTGGAC2501                          SerValLeuGlyAlaGluPheCysPheAspValThrPheGluValAsp                              730735740                                                                     ACGTCGGTTTTGGGCTGGGTGGTGGCCAGTGTGGTAGCCTGGGCCATT2549                          ThrSerValLeuGlyTrpValValAlaSerValValAlaTrpAlaIle                              745750755                                                                     GCGCTCCTGAGCTCGATGAGCGCGGGAGGGTGGAGGCACAAGGCCGTG2597                          AlaLeuLeuSerSerMetSerAlaGlyGlyTrpArgHisLysAlaVal                              760765770                                                                     ATCTATAGGACGTGGTGTAAGGGGTACCAGGCAATACGCCAACGGGTG2645                          IleTyrArgThrTrpCysLysGlyTyrGlnAlaIleArgGlnArgVal                              775780785790                                                                  GTGCGGAGCCCCCTCGGGGAGGGGCGGCCCACCAAACCCTTGACGTTT2693                          ValArgSerProLeuGlyGluGlyArgProThrLysProLeuThrPhe                              795800805                                                                     GCTTGGTGCTTGGCCTCATACATCTGGCCGGATGCTGTGATGATGGTG2741                          AlaTrpCysLeuAlaSerTyrIleTrpProAspAlaValMetMetVal                              810815820                                                                     GTGGTAGCCTTGGTGCTCCTCTTTGGCCTGTTCGACGCGTTGGACTGG2789                          ValValAlaLeuValLeuLeuPheGlyLeuPheAspAlaLeuAspTrp                              825830835                                                                     GCTTTGGAGGAGCTCTTGGTGTCCCGGCCCTCGTTACGGCGTCTGGCC2837                          AlaLeuGluGluLeuLeuValSerArgProSerLeuArgArgLeuAla                              840845850                                                                     CGGGTGGTTGAGTGCTGTGTGATGGCGGGAGAGAAGGCCACAACCGTC2885                          ArgValValGluCysCysValMetAlaGlyGluLysAlaThrThrVal                              855860865870                                                                  CGGCTGGTCTCCAAGATGTGCGCGAGAGGGGCCTATTTGTTTGACCAT2933                          ArgLeuValSerLysMetCysAlaArgGlyAlaTyrLeuPheAspHis                              875880885                                                                     ATGGGCTCTTTTTCGCGCGCTGTCAAGGAGCGCCTGCTGGAGTGGGAC2981                          MetGlySerPheSerArgAlaValLysGluArgLeuLeuGluTrpAsp                              890895900                                                                     GCGGCTTTGGAACCCCTGTCATTCACTAGGACGGACTGTCGCATCATT3029                          AlaAlaLeuGluProLeuSerPheThrArgThrAspCysArgIleIle                              905910915                                                                     AGAGATGCTGCGAGGACCTTGGCCTGCGGGCAGTGCGTCATGGGCTTG3077                          ArgAspAlaAlaArgThrLeuAlaCysGlyGlnCysValMetGlyLeu                              920925930                                                                     CCTGTGGTAGCGCGCCGTGGTGACGAGGTTCTTATCGGTGTCTTTCAG3125                          ProValValAlaArgArgGlyAspGluValLeuIleGlyValPheGln                              935940945950                                                                  GATGTGAACCATTTGCCTCCCGGATTCGTCCCGACCGCACCCGTTGTC3173                          AspValAsnHisLeuProProGlyPheValProThrAlaProValVal                              955960965                                                                     ATCCGGCGGTGCGGGAAGGGGTTTCTGGGGGTCACTAAGGCTGCCTTG3221                          IleArgArgCysGlyLysGlyPheLeuGlyValThrLysAlaAlaLeu                              970975980                                                                     ACTGGTCGGGATCCTGACTTACATCCAGGGAACGTCATGGTGTTGGGG3269                          ThrGlyArgAspProAspLeuHisProGlyAsnValMetValLeuGly                              985990995                                                                     ACGGCTACGTCGCGAAGCATGGGGACATGCCTGAACGGCCTGCTGTTC3317                          ThrAlaThrSerArgSerMetGlyThrCysLeuAsnGlyLeuLeuPhe                              100010051010                                                                  ACGACTTTCCATGGGGCTTCATCCCGAACCATCGCCACGCCCGTGGGG3365                          ThrThrPheHisGlyAlaSerSerArgThrIleAlaThrProValGly                              1015102010251030                                                              GCCCTTAATCCCAGGTGGTGGTCCGCCAGTGATGACGTCACGGTGTAC3413                          AlaLeuAsnProArgTrpTrpSerAlaSerAspAspValThrValTyr                              103510401045                                                                  CCGCTCCCGGATGGGGCAACCTCGTTGACGCCCTGCACTTGCCAGGCT3461                          ProLeuProAspGlyAlaThrSerLeuThrProCysThrCysGlnAla                              105010551060                                                                  GAGTCCTGTTGGGTCATACGGTCCGACGGGGCTTTGTGCCATGGCTTG3509                          GluSerCysTrpValIleArgSerAspGlyAlaLeuCysHisGlyLeu                              106510701075                                                                  AGTAAGGGAGACAAGGTGGAGCTAGATGTGGCCATGGAGGTCTCAGAT3557                          SerLysGlyAspLysValGluLeuAspValAlaMetGluValSerAsp                              108010851090                                                                  TTCCGTGGCTCGTCCGGCTCACCTGTCCTGTGCGACGAGGGGCACGCA3605                          PheArgGlySerSerGlySerProValLeuCysAspGluGlyHisAla                              1095110011051110                                                              GTAGGAATGCTCGTGTCGGTGCTCCACTCGGGTGGTCGGGTCACCGCG3653                          ValGlyMetLeuValSerValLeuHisSerGlyGlyArgValThrAla                              111511201125                                                                  GCTCGATTCACCAGGCCGTGGACCCAGGTCCCAACAGATGCTAAGACC3701                          AlaArgPheThrArgProTrpThrGlnValProThrAspAlaLysThr                              113011351140                                                                  ACCACTGAACCCCCTCCGGTGCCGGCAAAGGGAGTTTTCAAGGAAGCC3749                          ThrThrGluProProProValProAlaLysGlyValPheLysGluAla                              114511501155                                                                  CCACTGTTTATGCCCACGGGCGCAGGAAAGAGCACGCGCGTCCCGTTG3797                          ProLeuPheMetProThrGlyAlaGlyLysSerThrArgValProLeu                              116011651170                                                                  GAGTATGGCAACATGGGGCACAAGGTCCTGATTTTGAACCCCTCGGTG3845                          GluTyrGlyAsnMetGlyHisLysValLeuIleLeuAsnProSerVal                              1175118011851190                                                              GCGACAGTGAGGGCCATGGGCCCTTACATGGAGCGACTGGCGGGAAAA3893                          AlaThrValArgAlaMetGlyProTyrMetGluArgLeuAlaGlyLys                              119512001205                                                                  CATCCAAGTATCTACTGTGGCCATGACACCACTGCCTTCACAAGGATC3941                          HisProSerIleTyrCysGlyHisAspThrThrAlaPheThrArgIle                              121012151220                                                                  ACTGATTCCCCCTTAACGTACTCTACCTATGGGAGGTTTCTGGCCAAC3989                          ThrAspSerProLeuThrTyrSerThrTyrGlyArgPheLeuAlaAsn                              122512301235                                                                  CCTAGGCAGATGCTGCGAGGTGTGTCGGTGGTCATTTGCGATGAATGC4037                          ProArgGlnMetLeuArgGlyValSerValValIleCysAspGluCys                              124012451250                                                                  CACAGTCATGATTCCACTGTGTTGTTGGGGATTGGACGGGTCCGGGAG4085                          HisSerHisAspSerThrValLeuLeuGlyIleGlyArgValArgGlu                              1255126012651270                                                              CTGGCACGAGAGTGTGGGGTGCAGCTTGTGCTCTACGCCACTGCCACG4133                          LeuAlaArgGluCysGlyValGlnLeuValLeuTyrAlaThrAlaThr                              127512801285                                                                  CCTCCTGGGTCCCCCATGACTCAGCATCCGTCAATCATTGAGACCAAA4181                          ProProGlySerProMetThrGlnHisProSerIleIleGluThrLys                              129012951300                                                                  TTGGATGTGGGTGAGATTCCCTTCTATGGGCATGGCATACCCCTCGAG4229                          LeuAspValGlyGluIleProPheTyrGlyHisGlyIleProLeuGlu                              130513101315                                                                  CGGATGCGGACCGGTAGGCACCTCGTATTCTGCTACTCTAAGGCAGAG4277                          ArgMetArgThrGlyArgHisLeuValPheCysTyrSerLysAlaGlu                              132013251330                                                                  TGTGAGCGGCTAGCCGGTCAGTTTTCTGCTAGGGGAGTTAACGCCATA4325                          CysGluArgLeuAlaGlyGlnPheSerAlaArgGlyValAsnAlaIle                              1335134013451350                                                              GCCTATTACAGGGGAAAAGACAGTTCTATCATCAAGGACGGAGATCTG4373                          AlaTyrTyrArgGlyLysAspSerSerIleIleLysAspGlyAspLeu                              135513601365                                                                  GTGGTGTGCGCGACCGACGCGCTATCCACTGGATACACTGGGAACTTC4421                          ValValCysAlaThrAspAlaLeuSerThrGlyTyrThrGlyAsnPhe                              137013751380                                                                  GATTCTGTCACCGACTGTGGGTTAGTGGTGGAGGAGGTCGTCGAGGTG4469                          AspSerValThrAspCysGlyLeuValValGluGluValValGluVal                              138513901395                                                                  ACCCTTGATCCCACCATTACCATCTCCCTGCGGACAGTGCCCGCGTCG4517                          ThrLeuAspProThrIleThrIleSerLeuArgThrValProAlaSer                              140014051410                                                                  GCAGAACTGTCGATGCAGAGACGAGGACGCACGGGTAGAGGCAGGTCT4565                          AlaGluLeuSerMetGlnArgArgGlyArgThrGlyArgGlyArgSer                              1415142014251430                                                              GGGCGCTACTACTACGCCGGGGTCGGAAAGGCCCCCGCGGGTGTGGTG4613                          GlyArgTyrTyrTyrAlaGlyValGlyLysAlaProAlaGlyValVal                              143514401445                                                                  CGCTCGGGTCCTGTCTGGTCGGCGGTGGAGGCCGGAGTGACCTGGTAT4661                          ArgSerGlyProValTrpSerAlaValGluAlaGlyValThrTrpTyr                              145014551460                                                                  GGAATGGAACCTGACTTGACAGCTAACCTATTGAGACTTTACGACGAC4709                          GlyMetGluProAspLeuThrAlaAsnLeuLeuArgLeuTyrAspAsp                              146514701475                                                                  TGCCCTTACACCGCAGCCGTCGCAGCTGACATCGGTGAAGCCGCGGTG4757                          CysProTyrThrAlaAlaValAlaAlaAspIleGlyGluAlaAlaVal                              148014851490                                                                  TTTTTCTCCGGGCTAGCCCCGTTGAGGATGCATCCCGATGTTAGCTGG4805                          PhePheSerGlyLeuAlaProLeuArgMetHisProAspValSerTrp                              1495150015051510                                                              GCAAAAGTGCGCGGCGTCAACTGGCCCCTCTTGGTGGGTGTTCAGCGG4853                          AlaLysValArgGlyValAsnTrpProLeuLeuValGlyValGlnArg                              151515201525                                                                  ACCATGTGCCGGGAAACACTGTCTCCCGGACCATCGGACGACCCCCAA4901                          ThrMetCysArgGluThrLeuSerProGlyProSerAspAspProGln                              153015351540                                                                  TGGGCAGGTCTGAAGGGCCCGAATCCTGTTCCACTACTGCTGAGGTGG4949                          TrpAlaGlyLeuLysGlyProAsnProValProLeuLeuLeuArgTrp                              154515501555                                                                  GGCAATGATTTACCATCAAAAGTGGCCGGCCACCACATTGTTGACGAC4997                          GlyAsnAspLeuProSerLysValAlaGlyHisHisIleValAspAsp                              156015651570                                                                  CTGGTTCGTAGGCTTGGTGTGGCGGAGGGTTATGTCCGCTGCGATGCG5045                          LeuValArgArgLeuGlyValAlaGluGlyTyrValArgCysAspAla                              1575158015851590                                                              GGGCCGATCTTAATGGTCGGCCTCGCTATCGCGGGGGGGATGATCTAC5093                          GlyProIleLeuMetValGlyLeuAlaIleAlaGlyGlyMetIleTyr                              159516001605                                                                  GCATCTTACACCGGGTCTTTAGTGGTGGTGACAGACTGGGATGTAAAG5141                          AlaSerTyrThrGlySerLeuValValValThrAspTrpAspValLys                              161016151620                                                                  GGGGGTGGCAGCCCTCTTTATCGGCATGGAGACCAGGCCACGCCACAG5189                          GlyGlyGlySerProLeuTyrArgHisGlyAspGlnAlaThrProGln                              162516301635                                                                  CCGGTTGTGCAGGTCCCCCCGGTAGACCATCGGCCGGGGGGGGAGTCT5237                          ProValValGlnValProProValAspHisArgProGlyGlyGluSer                              164016451650                                                                  GCGCCTTCGGATGCCAAGACAGTGACAGATGCGGTGGCGGCCATCCAG5285                          AlaProSerAspAlaLysThrValThrAspAlaValAlaAlaIleGln                              1655166016651670                                                              GTGGATTGCGATTGGTCAGTCATGACCCTGTCGATCGGGGAAGTGCTG5333                          ValAspCysAspTrpSerValMetThrLeuSerIleGlyGluValLeu                              167516801685                                                                  TCCTTGGCTCAGGCTAAAACAGCTGAGGCCTACACGGCAACCGCCAAG5381                          SerLeuAlaGlnAlaLysThrAlaGluAlaTyrThrAlaThrAlaLys                              169016951700                                                                  TGGCTCGCTGGCTGCTACACGGGGACGCGGGCCGTTCCCACTGTTTCA5429                          TrpLeuAlaGlyCysTyrThrGlyThrArgAlaValProThrValSer                              170517101715                                                                  ATTGTTGACAAGCTCTTTGCCGGAGGGTGGGCGGCTGTGGTTGGCCAC5477                          IleValAspLysLeuPheAlaGlyGlyTrpAlaAlaValValGlyHis                              172017251730                                                                  TGTCACAGCGTCATAGCTGCGGCGGTGGCTGCCTACGGGGCTTCCAGG5525                          CysHisSerValIleAlaAlaAlaValAlaAlaTyrGlyAlaSerArg                              1735174017451750                                                              AGTCCGCCGTTGGCAGCCGCGGCTTCCTACCTGATGGGACTGGGCGTC5573                          SerProProLeuAlaAlaAlaAlaSerTyrLeuMetGlyLeuGlyVal                              175517601765                                                                  GGAGGCAACGCTCAGACGCGTTTGGCGTCTGCCCTCCTGTTGGGGGCC5621                          GlyGlyAsnAlaGlnThrArgLeuAlaSerAlaLeuLeuLeuGlyAla                              177017751780                                                                  GCTGGCACCGCCCTGGGCACTCCCGTCGTGGGTTTAACCATGGCGGGG5669                          AlaGlyThrAlaLeuGlyThrProValValGlyLeuThrMetAlaGly                              178517901795                                                                  GCGTTCATGGGGGGTGCTAGCGTCTCTCCCTCCTTGGTCACCATCTTG5717                          AlaPheMetGlyGlyAlaSerValSerProSerLeuValThrIleLeu                              180018051810                                                                  TTGGGGGCCGTGGGAGGCTGGGAGGGCGTCGTCAACGCTGCTAGCCTT5765                          LeuGlyAlaValGlyGlyTrpGluGlyValValAsnAlaAlaSerLeu                              1815182018251830                                                              GTCTTTGACTTCATGGCGGGGAAACTATCGTCAGAAGATCTGTGGTAC5813                          ValPheAspPheMetAlaGlyLysLeuSerSerGluAspLeuTrpTyr                              183518401845                                                                  GCCATCCCAGTGCTCACCAGCCCGGGGGCGGGCCTTGCGGGGATCGCC5861                          AlaIleProValLeuThrSerProGlyAlaGlyLeuAlaGlyIleAla                              185018551860                                                                  CTTGGGTTGGTGCTGTACTCAGCTAACAACTCTGGTACTACCACTTGG5909                          LeuGlyLeuValLeuTyrSerAlaAsnAsnSerGlyThrThrThrTrp                              186518701875                                                                  TTGAACCGTCTGCTGACTACGTTACCTAGGTCTTCTTGCATCCCTGAC5957                          LeuAsnArgLeuLeuThrThrLeuProArgSerSerCysIleProAsp                              188018851890                                                                  AGCTATTTCCAACAGGCCGATTACTGTGACAAGGTCTCGGCCGTGCTT6005                          SerTyrPheGlnGlnAlaAspTyrCysAspLysValSerAlaValLeu                              1895190019051910                                                              CGCCGACTGAGCCTCACCCGCACTGTGGTGGCCCTAGTCAATAGGGAA6053                          ArgArgLeuSerLeuThrArgThrValValAlaLeuValAsnArgGlu                              191519201925                                                                  CCCAAGGTGGACGAGGTACAGGTGGGGTACGTCTGGGATCTCTGGGAG6101                          ProLysValAspGluValGlnValGlyTyrValTrpAspLeuTrpGlu                              193019351940                                                                  TGGATCATGCGTCAAGTGCGCATGGTCATGGCCAGGCTCCGGGCTCTC6149                          TrpIleMetArgGlnValArgMetValMetAlaArgLeuArgAlaLeu                              194519501955                                                                  TGCCCCGTGGTGTCACTGCCTTTGTGGCACTGCGGGGAGGGGTGGTCC6197                          CysProValValSerLeuProLeuTrpHisCysGlyGluGlyTrpSer                              196019651970                                                                  GGAGAGTGGTTGTTGGACGGCCATGTGGAGAGTCGCTGTCTTTGCGGG6245                          GlyGluTrpLeuLeuAspGlyHisValGluSerArgCysLeuCysGly                              1975198019851990                                                              TGCGTGATCACCGGCGATGTTTTCAATGGGCAACTCAAAGAGCCAGTT6293                          CysValIleThrGlyAspValPheAsnGlyGlnLeuLysGluProVal                              199520002005                                                                  TACTCTACAAAGTTGTGCCGGCACTATTGGATGGGGACCGTTCCTGTG6341                          TyrSerThrLysLeuCysArgHisTyrTrpMetGlyThrValProVal                              201020152020                                                                  AACATGCTGGGTTACGGCGAAACATCACCCCTCTTGGCCTCTGACACC6389                          AsnMetLeuGlyTyrGlyGluThrSerProLeuLeuAlaSerAspThr                              202520302035                                                                  CCGAAGGTGGTGCCTTTTGGGACGTCGGGCTGGGCTGAGGTGGTGGTG6437                          ProLysValValProPheGlyThrSerGlyTrpAlaGluValValVal                              204020452050                                                                  ACCCCTACCCACGTGGTGATCAGGAGAACCTCTCCCTACGAGTTGCTG6485                          ThrProThrHisValValIleArgArgThrSerProTyrGluLeuLeu                              2055206020652070                                                              CGCCAACAAATCCTATCAGCTGCAGTTGCTGAGCCCTATTATGTCGAC6533                          ArgGlnGlnIleLeuSerAlaAlaValAlaGluProTyrTyrValAsp                              207520802085                                                                  GGCATACCGGTCTCATGGGACGCGGACGCTCGTGCGCCTGCTATGGTT6581                          GlyIleProValSerTrpAspAlaAspAlaArgAlaProAlaMetVal                              209020952100                                                                  TATGGCCCTGGGCAAAGTGTTACCATTGACGGGGAGCGCTACACCCTG6629                          TyrGlyProGlyGlnSerValThrIleAspGlyGluArgTyrThrLeu                              210521102115                                                                  CCGCATCAACTGCGGCTCAGGAATGTAGCGCCCTCTGAGGTTTCATCC6677                          ProHisGlnLeuArgLeuArgAsnValAlaProSerGluValSerSer                              212021252130                                                                  GAGGTGTCCATAGACATTGGGACGGAGACTGAAGACTCAGAACTGACT6725                          GluValSerIleAspIleGlyThrGluThrGluAspSerGluLeuThr                              2135214021452150                                                              GAGGCCGACCTGCCGCCGGCAGCTGCAGCCCTCCAGGCTATCGAGAAT6773                          GluAlaAspLeuProProAlaAlaAlaAlaLeuGlnAlaIleGluAsn                              215521602165                                                                  GCTGCGAGGATTCTTGAGCCTCATATTGATGTCATCATGGAGGATTGC6821                          AlaAlaArgIleLeuGluProHisIleAspValIleMetGluAspCys                              217021752180                                                                  AGTACACCCTCTCTTTGTGGTAGTAGCCGAGAGATGCCTGTGTGGGGA6869                          SerThrProSerLeuCysGlySerSerArgGluMetProValTrpGly                              218521902195                                                                  GAAGACATCCCCCGCACTCCATCGCCAGCACTTATCTCGGTTACCGAG6917                          GluAspIleProArgThrProSerProAlaLeuIleSerValThrGlu                              220022052210                                                                  AGCAGCTCAGATGAGAAGACCCCGTCGGTGTCCTCCTCGCAGGAGGAT6965                          SerSerSerAspGluLysThrProSerValSerSerSerGlnGluAsp                              2215222022252230                                                              ACCCCGTCCTCTGACTCATTCGAAGTCATCCAAGAGTCTGAGACAGCT7013                          ThrProSerSerAspSerPheGluValIleGlnGluSerGluThrAla                              223522402245                                                                  GAAGGAGAGGAAAGTGTCTTCAACGTGGCTCTTTCCGTACTAGAAGCC7061                          GluGlyGluGluSerValPheAsnValAlaLeuSerValLeuGluAla                              225022552260                                                                  TTGTTTCCACAGAGTGATGCCACTAGAAAGCTTACCGTCAGGATGAAT7109                          LeuPheProGlnSerAspAlaThrArgLysLeuThrValArgMetAsn                              226522702275                                                                  TGCTGCGTTGAGAAGAGCGTCACGCGCTTCTTTTCTTTGGGGCTGACG7157                          CysCysValGluLysSerValThrArgPhePheSerLeuGlyLeuThr                              228022852290                                                                  GTGGCTGATGTGGCCAGTCTGTGTGAGATGGAGATCCAGAACCATACA7205                          ValAlaAspValAlaSerLeuCysGluMetGluIleGlnAsnHisThr                              2295230023052310                                                              GCCTATTGTGACAAGGTGCGCACTCCGCTCGAATTGCAAGTTGGGTGC7253                          AlaTyrCysAspLysValArgThrProLeuGluLeuGlnValGlyCys                              231523202325                                                                  TTGGTGGGCAATGAACTTACCTTTGAATGTGATAAGTGTGAGGCTAGG7301                          LeuValGlyAsnGluLeuThrPheGluCysAspLysCysGluAlaArg                              233023352340                                                                  CAAGAGACTTTGGCCTCCTTCTCCTATATTTGGTCTGGGGTGCCATTG7349                          GlnGluThrLeuAlaSerPheSerTyrIleTrpSerGlyValProLeu                              234523502355                                                                  ACTAGGGCCACACCGGCTAAACCACCTGTGGTGAGGCCGGTGGGGTCC7397                          ThrArgAlaThrProAlaLysProProValValArgProValGlySer                              236023652370                                                                  TTGTTGGTGGCTGACACCACGAAAGTGTATGTCACAAACCCGGACAAT7445                          LeuLeuValAlaAspThrThrLysValTyrValThrAsnProAspAsn                              2375238023852390                                                              GTTGGGAGAAGAGTGGACAAGGTGACCTTCTGGCGCGCCCCCAGGGTC7493                          ValGlyArgArgValAspLysValThrPheTrpArgAlaProArgVal                              239524002405                                                                  CATGACAAATATCTCGTGGACTCCATCGAGCGTGCCAGGAGGGCGGCT7541                          HisAspLysTyrLeuValAspSerIleGluArgAlaArgArgAlaAla                              241024152420                                                                  CAAGCCTGCCAAAGCATGGGTTACACTTATGAGGAAGCAATAAGGACT7589                          GlnAlaCysGlnSerMetGlyTyrThrTyrGluGluAlaIleArgThr                              242524302435                                                                  GTTAGGCCACATGCTGCCATGGGCTGGGGATCTAAGGTGTCGGTCAAG7637                          ValArgProHisAlaAlaMetGlyTrpGlySerLysValSerValLys                              244024452450                                                                  GACTTGGCCACCCCTGCGGGGAAGATGGCCGTCCACGACCGACTTCAG7685                          AspLeuAlaThrProAlaGlyLysMetAlaValHisAspArgLeuGln                              2455246024652470                                                              GAGATACTTGAGGGGACTCCGGTCCCTTTTACTCTTACTGTGAAAAAG7733                          GluIleLeuGluGlyThrProValProPheThrLeuThrValLysLys                              247524802485                                                                  GAGGTGTTCTTCAAAGACCGTAAGGAGGAGAAGGCCCCCCGCCTCATT7781                          GluValPhePheLysAspArgLysGluGluLysAlaProArgLeuIle                              249024952500                                                                  GTGTTCCCCCCCCTGGACTTCCGGATAGCTGAGAAGCTTATCCTGGGA7829                          ValPheProProLeuAspPheArgIleAlaGluLysLeuIleLeuGly                              250525102515                                                                  GACCCGGGGCGGGTGGCCAAGGCGGTGTTGGGGGGGGCTTACGCCTTC7877                          AspProGlyArgValAlaLysAlaValLeuGlyGlyAlaTyrAlaPhe                              252025252530                                                                  CAGTACACCCCAAATCAGCGAGTTAAGGAGATGCTCAAACTGTGGGAG7925                          GlnTyrThrProAsnGlnArgValLysGluMetLeuLysLeuTrpGlu                              2535254025452550                                                              TCAAAGAAAACACCTTGCGCCATCTGTGTGGACGCCACTTGCTTCGAC7973                          SerLysLysThrProCysAlaIleCysValAspAlaThrCysPheAsp                              255525602565                                                                  AGTAGCATTACTGAAGAGGACGTGGCGCTGGAGACAGAGCTGTACGCT8021                          SerSerIleThrGluGluAspValAlaLeuGluThrGluLeuTyrAla                              257025752580                                                                  CTGGCCTCTGACCATCCAGAGTGGGTGCGAGCTTTGGGGAAGTACTAT8069                          LeuAlaSerAspHisProGluTrpValArgAlaLeuGlyLysTyrTyr                              258525902595                                                                  GCCTCAGGAACCATGGTCACCCCTGAGGGGGTTCCCGTAGGTGAGAGG8117                          AlaSerGlyThrMetValThrProGluGlyValProValGlyGluArg                              260026052610                                                                  TATTGTAGATCCTCAGGCGTTTTGACTACCAGCGCGAGTAACTGCCTG8165                          TyrCysArgSerSerGlyValLeuThrThrSerAlaSerAsnCysLeu                              2615262026252630                                                              ACCTGCTACATCAAGGTGAAAGCCGCTTGTGAGAGAGTGGGGCTGAAA8213                          ThrCysTyrIleLysValLysAlaAlaCysGluArgValGlyLeuLys                              263526402645                                                                  AATGTCTCGCTTCTCATAGCCGGCGATGACTGTTTGATCATATGCGAA8261                          AsnValSerLeuLeuIleAlaGlyAspAspCysLeuIleIleCysGlu                              265026552660                                                                  CGGCCAGTGTGCGACCCTTGTGACGCCTTGGGCAGAGCCCTGGCGAGC8309                          ArgProValCysAspProCysAspAlaLeuGlyArgAlaLeuAlaSer                              266526702675                                                                  TATGGGTATGCTTGCGAGCCTTCGTATCATGCATCACTGGACACGGCC8357                          TyrGlyTyrAlaCysGluProSerTyrHisAlaSerLeuAspThrAla                              268026852690                                                                  CCCTTCTGCTCCACTTGGCTCGCTGAGTGCAACGCAGATGGGAAACGC8405                          ProPheCysSerThrTrpLeuAlaGluCysAsnAlaAspGlyLysArg                              2695270027052710                                                              CATTTCTTCCTGACCACGGACTTTCGGAGGCCGCTTGCTCGCATGTCG8453                          HisPhePheLeuThrThrAspPheArgArgProLeuAlaArgMetSer                              271527202725                                                                  AGCGAGTATAGTGACCCAATGGCTTCGGCCATAGGTTACATCCTCCTG8501                          SerGluTyrSerAspProMetAlaSerAlaIleGlyTyrIleLeuLeu                              273027352740                                                                  TATCCCTGGCATCCCATCACACGGTGGGTCATCATCCCTCATGTGCTA8549                          TyrProTrpHisProIleThrArgTrpValIleIleProHisValLeu                              274527502755                                                                  ACGTGCGCATTCAGGGGTGGTGGTACACCGTCTGATCCGGTTTGGTGT8597                          ThrCysAlaPheArgGlyGlyGlyThrProSerAspProValTrpCys                              276027652770                                                                  CAGGTGCATGGTAACTACTACAAGTTTCCACTGGACAAACTGCCTAAC8645                          GlnValHisGlyAsnTyrTyrLysPheProLeuAspLysLeuProAsn                              2775278027852790                                                              ATCATCGTGGCCCTCCACGGACCAGCAGCGTTGAGGGTTACCGCAGAC8693                          IleIleValAlaLeuHisGlyProAlaAlaLeuArgValThrAlaAsp                              279528002805                                                                  ACAACTAAGACAAAAATGGAAGCTGGGAAGGTGCTGAGTGACCTCAAG8741                          ThrThrLysThrLysMetGluAlaGlyLysValLeuSerAspLeuLys                              281028152820                                                                  CTCCCTGGCCTAGCGGTCCACCGAAAGAAGGCCGGAGCACTGCGAACA8789                          LeuProGlyLeuAlaValHisArgLysLysAlaGlyAlaLeuArgThr                              282528302835                                                                  CGCATGCTTCGGTCGCGCGGTTGGGCCGAGTTGGCGAGGGGCCTGTTG8837                          ArgMetLeuArgSerArgGlyTrpAlaGluLeuAlaArgGlyLeuLeu                              284028452850                                                                  TGGCATCCAGGCCTCCGGCTCCCTCCCCCTGAGATTGCTGGTATCCCG8885                          TrpHisProGlyLeuArgLeuProProProGluIleAlaGlyIlePro                              2855286028652870                                                              GGGGGTTTCCCCCTCTCCCCCCCCTACATGGGGGTGGTGCATCAATTG8933                          GlyGlyPheProLeuSerProProTyrMetGlyValValHisGlnLeu                              287528802885                                                                  GATTTTACAAGCCAGAGGAGTCGCTGGCGGTGGCTGGGGTTCTTAGCC8981                          AspPheThrSerGlnArgSerArgTrpArgTrpLeuGlyPheLeuAla                              289028952900                                                                  CTGCTCATCGTAGCCCTCTTCGGGTGAACTAAATTCATCTGTTGCGGCAAGGTC9035                    LeuLeuIleValAlaLeuPheGly                                                      29052910                                                                      CAGTGACTGATCATCACTGGAGGAGGTTCCCGCCCTCCCCGCCCCAGGGGTCTCCCCGCT9095              GGGTAAAA9103                                                                  (2) INFORMATION FOR SEQ ID NO:183:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2910 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:                                     MetSerLeuLeuThrAsnArgLeuSerArgArgValAspLysAspGln                              151015                                                                        TrpGlyProGlyPheMetGlyLysAspProLysProCysProSerArg                              202530                                                                        ArgThrGlyLysCysMetGlyProProSerSerAlaAlaAlaCysSer                              354045                                                                        ArgGlySerProArgIleLeuArgValArgAlaGlyGlyIleSerLeu                              505560                                                                        ProTyrThrIleMetGluAlaLeuLeuPheLeuLeuGlyValGluAla                              65707580                                                                      GlyAlaIleLeuAlaProAlaThrHisAlaCysArgAlaAsnGlyGln                              859095                                                                        TyrPheLeuThrAsnCysCysAlaProGluAspIleGlyPheCysLeu                              100105110                                                                     GluGlyGlyCysLeuValAlaLeuGlyCysThrValCysThrAspArg                              115120125                                                                     CysTrpProLeuTyrGlnAlaGlyLeuAlaValArgProGlyLysSer                              130135140                                                                     AlaAlaGlnLeuValGlyGlnLeuGlyGlyLeuTyrGlyProLeuSer                              145150155160                                                                  ValSerAlaTyrValAlaGlyIleLeuGlyLeuGlyGluValTyrSer                              165170175                                                                     GlyValLeuThrValGlyValAlaLeuThrArgArgValTyrProMet                              180185190                                                                     ProAsnLeuThrCysAlaValGluCysGluLeuLysTrpGluSerGlu                              195200205                                                                     PheTrpArgTrpThrGluGlnLeuAlaSerAsnTyrTrpIleLeuGlu                              210215220                                                                     TyrLeuTrpLysValProPheAspPheTrpArgGlyValLeuSerLeu                              225230235240                                                                  ThrProLeuLeuValCysValAlaAlaLeuLeuLeuLeuGluGlnArg                              245250255                                                                     IleValMetValPheLeuLeuValThrMetAlaGlyMetSerGlnGly                              260265270                                                                     AlaProAlaSerValLeuGlySerArgProPheAspTyrGlyLeuThr                              275280285                                                                     TrpGlnSerCysSerCysArgAlaAsnGlySerArgTyrThrThrGly                              290295300                                                                     GluLysValTrpAspArgGlyAsnValThrLeuLeuCysAspCysPro                              305310315320                                                                  AsnGlyProTrpValTrpLeuProAlaPheCysGlnAlaIleGlyTrp                              325330335                                                                     GlyAspProIleThrHisTrpSerHisGlyGlnAsnArgTrpProLeu                              340345350                                                                     SerCysProGlnTyrValTyrGlySerValSerValThrCysValTrp                              355360365                                                                     GlySerValSerTrpPheAlaSerThrGlyGlyArgAspSerLysIle                              370375380                                                                     AspValTrpSerLeuValProValGlySerAlaSerCysThrIleAla                              385390395400                                                                  AlaLeuGlySerSerAspArgAspThrValValGluLeuSerGluTrp                              405410415                                                                     GlyValProCysAlaThrCysIleLeuAspArgArgProAlaSerCys                              420425430                                                                     GlyThrCysValArgAspCysTrpProGluThrGlySerValArgPhe                              435440445                                                                     ProPheHisArgCysGlyAlaGlyProLysLeuThrLysAspLeuGlu                              450455460                                                                     AlaValProPheValAsnArgThrThrProPheThrIleArgGlyPro                              465470475480                                                                  LeuGlyAsnGlnGlyArgGlyAsnProValArgSerProLeuGlyPhe                              485490495                                                                     GlySerTyrAlaMetThrLysIleArgAspSerLeuHisLeuValLys                              500505510                                                                     CysProThrProAlaIleGluProProThrGlyThrPheGlyPhePhe                              515520525                                                                     ProGlyValProProLeuAsnAsnCysLeuLeuLeuGlyThrGluVal                              530535540                                                                     SerGluAlaLeuGlyGlyAlaGlyLeuThrGlyGlyPheTyrGluPro                              545550555560                                                                  LeuValArgArgArgSerGluLeuMetGlyArgArgAsnProValCys                              565570575                                                                     ProGlyPheAlaTrpLeuSerSerGlyArgProAspGlyPheIleHis                              580585590                                                                     ValGlnGlyHisLeuGlnGluValAspAlaGlyAsnPheIleProPro                              595600605                                                                     ProArgTrpLeuLeuLeuAspPheValPheValLeuLeuTyrLeuMet                              610615620                                                                     LysLeuAlaGluAlaArgLeuValProLeuIleLeuLeuLeuLeuTrp                              625630635640                                                                  TrpTrpValAsnGlnLeuAlaValLeuGlyLeuProAlaValAspAla                              645650655                                                                     AlaValAlaGlyGluValPheAlaGlyProAlaLeuSerTrpCysLeu                              660665670                                                                     GlyLeuProThrValSerMetIleLeuGlyLeuAlaAsnLeuValLeu                              675680685                                                                     TyrPheArgTrpMetGlyProGlnArgLeuMetPheLeuValLeuTrp                              690695700                                                                     LysLeuAlaArgGlyAlaPheProLeuAlaLeuLeuMetGlyIleSer                              705710715720                                                                  AlaThrArgGlyArgThrSerValLeuGlyAlaGluPheCysPheAsp                              725730735                                                                     ValThrPheGluValAspThrSerValLeuGlyTrpValValAlaSer                              740745750                                                                     ValValAlaTrpAlaIleAlaLeuLeuSerSerMetSerAlaGlyGly                              755760765                                                                     TrpArgHisLysAlaValIleTyrArgThrTrpCysLysGlyTyrGln                              770775780                                                                     AlaIleArgGlnArgValValArgSerProLeuGlyGluGlyArgPro                              785790795800                                                                  ThrLysProLeuThrPheAlaTrpCysLeuAlaSerTyrIleTrpPro                              805810815                                                                     AspAlaValMetMetValValValAlaLeuValLeuLeuPheGlyLeu                              820825830                                                                     PheAspAlaLeuAspTrpAlaLeuGluGluLeuLeuValSerArgPro                              835840845                                                                     SerLeuArgArgLeuAlaArgValValGluCysCysValMetAlaGly                              850855860                                                                     GluLysAlaThrThrValArgLeuValSerLysMetCysAlaArgGly                              865870875880                                                                  AlaTyrLeuPheAspHisMetGlySerPheSerArgAlaValLysGlu                              885890895                                                                     ArgLeuLeuGluTrpAspAlaAlaLeuGluProLeuSerPheThrArg                              900905910                                                                     ThrAspCysArgIleIleArgAspAlaAlaArgThrLeuAlaCysGly                              915920925                                                                     GlnCysValMetGlyLeuProValValAlaArgArgGlyAspGluVal                              930935940                                                                     LeuIleGlyValPheGlnAspValAsnHisLeuProProGlyPheVal                              945950955960                                                                  ProThrAlaProValValIleArgArgCysGlyLysGlyPheLeuGly                              965970975                                                                     ValThrLysAlaAlaLeuThrGlyArgAspProAspLeuHisProGly                              980985990                                                                     AsnValMetValLeuGlyThrAlaThrSerArgSerMetGlyThrCys                              99510001005                                                                   LeuAsnGlyLeuLeuPheThrThrPheHisGlyAlaSerSerArgThr                              101010151020                                                                  IleAlaThrProValGlyAlaLeuAsnProArgTrpTrpSerAlaSer                              1025103010351040                                                              AspAspValThrValTyrProLeuProAspGlyAlaThrSerLeuThr                              104510501055                                                                  ProCysThrCysGlnAlaGluSerCysTrpValIleArgSerAspGly                              106010651070                                                                  AlaLeuCysHisGlyLeuSerLysGlyAspLysValGluLeuAspVal                              107510801085                                                                  AlaMetGluValSerAspPheArgGlySerSerGlySerProValLeu                              109010951100                                                                  CysAspGluGlyHisAlaValGlyMetLeuValSerValLeuHisSer                              1105111011151120                                                              GlyGlyArgValThrAlaAlaArgPheThrArgProTrpThrGlnVal                              112511301135                                                                  ProThrAspAlaLysThrThrThrGluProProProValProAlaLys                              114011451150                                                                  GlyValPheLysGluAlaProLeuPheMetProThrGlyAlaGlyLys                              115511601165                                                                  SerThrArgValProLeuGluTyrGlyAsnMetGlyHisLysValLeu                              117011751180                                                                  IleLeuAsnProSerValAlaThrValArgAlaMetGlyProTyrMet                              1185119011951200                                                              GluArgLeuAlaGlyLysHisProSerIleTyrCysGlyHisAspThr                              120512101215                                                                  ThrAlaPheThrArgIleThrAspSerProLeuThrTyrSerThrTyr                              122012251230                                                                  GlyArgPheLeuAlaAsnProArgGlnMetLeuArgGlyValSerVal                              123512401245                                                                  ValIleCysAspGluCysHisSerHisAspSerThrValLeuLeuGly                              125012551260                                                                  IleGlyArgValArgGluLeuAlaArgGluCysGlyValGlnLeuVal                              1265127012751280                                                              LeuTyrAlaThrAlaThrProProGlySerProMetThrGlnHisPro                              128512901295                                                                  SerIleIleGluThrLysLeuAspValGlyGluIleProPheTyrGly                              130013051310                                                                  HisGlyIleProLeuGluArgMetArgThrGlyArgHisLeuValPhe                              131513201325                                                                  CysTyrSerLysAlaGluCysGluArgLeuAlaGlyGlnPheSerAla                              133013351340                                                                  ArgGlyValAsnAlaIleAlaTyrTyrArgGlyLysAspSerSerIle                              1345135013551360                                                              IleLysAspGlyAspLeuValValCysAlaThrAspAlaLeuSerThr                              136513701375                                                                  GlyTyrThrGlyAsnPheAspSerValThrAspCysGlyLeuValVal                              138013851390                                                                  GluGluValValGluValThrLeuAspProThrIleThrIleSerLeu                              139514001405                                                                  ArgThrValProAlaSerAlaGluLeuSerMetGlnArgArgGlyArg                              141014151420                                                                  ThrGlyArgGlyArgSerGlyArgTyrTyrTyrAlaGlyValGlyLys                              1425143014351440                                                              AlaProAlaGlyValValArgSerGlyProValTrpSerAlaValGlu                              144514501455                                                                  AlaGlyValThrTrpTyrGlyMetGluProAspLeuThrAlaAsnLeu                              146014651470                                                                  LeuArgLeuTyrAspAspCysProTyrThrAlaAlaValAlaAlaAsp                              147514801485                                                                  IleGlyGluAlaAlaValPhePheSerGlyLeuAlaProLeuArgMet                              149014951500                                                                  HisProAspValSerTrpAlaLysValArgGlyValAsnTrpProLeu                              1505151015151520                                                              LeuValGlyValGlnArgThrMetCysArgGluThrLeuSerProGly                              152515301535                                                                  ProSerAspAspProGlnTrpAlaGlyLeuLysGlyProAsnProVal                              154015451550                                                                  ProLeuLeuLeuArgTrpGlyAsnAspLeuProSerLysValAlaGly                              155515601565                                                                  HisHisIleValAspAspLeuValArgArgLeuGlyValAlaGluGly                              157015751580                                                                  TyrValArgCysAspAlaGlyProIleLeuMetValGlyLeuAlaIle                              1585159015951600                                                              AlaGlyGlyMetIleTyrAlaSerTyrThrGlySerLeuValValVal                              160516101615                                                                  ThrAspTrpAspValLysGlyGlyGlySerProLeuTyrArgHisGly                              162016251630                                                                  AspGlnAlaThrProGlnProValValGlnValProProValAspHis                              163516401645                                                                  ArgProGlyGlyGluSerAlaProSerAspAlaLysThrValThrAsp                              165016551660                                                                  AlaValAlaAlaIleGlnValAspCysAspTrpSerValMetThrLeu                              1665167016751680                                                              SerIleGlyGluValLeuSerLeuAlaGlnAlaLysThrAlaGluAla                              168516901695                                                                  TyrThrAlaThrAlaLysTrpLeuAlaGlyCysTyrThrGlyThrArg                              170017051710                                                                  AlaValProThrValSerIleValAspLysLeuPheAlaGlyGlyTrp                              171517201725                                                                  AlaAlaValValGlyHisCysHisSerValIleAlaAlaAlaValAla                              173017351740                                                                  AlaTyrGlyAlaSerArgSerProProLeuAlaAlaAlaAlaSerTyr                              1745175017551760                                                              LeuMetGlyLeuGlyValGlyGlyAsnAlaGlnThrArgLeuAlaSer                              176517701775                                                                  AlaLeuLeuLeuGlyAlaAlaGlyThrAlaLeuGlyThrProValVal                              178017851790                                                                  GlyLeuThrMetAlaGlyAlaPheMetGlyGlyAlaSerValSerPro                              179518001805                                                                  SerLeuValThrIleLeuLeuGlyAlaValGlyGlyTrpGluGlyVal                              181018151820                                                                  ValAsnAlaAlaSerLeuValPheAspPheMetAlaGlyLysLeuSer                              1825183018351840                                                              SerGluAspLeuTrpTyrAlaIleProValLeuThrSerProGlyAla                              184518501855                                                                  GlyLeuAlaGlyIleAlaLeuGlyLeuValLeuTyrSerAlaAsnAsn                              186018651870                                                                  SerGlyThrThrThrTrpLeuAsnArgLeuLeuThrThrLeuProArg                              187518801885                                                                  SerSerCysIleProAspSerTyrPheGlnGlnAlaAspTyrCysAsp                              189018951900                                                                  LysValSerAlaValLeuArgArgLeuSerLeuThrArgThrValVal                              1905191019151920                                                              AlaLeuValAsnArgGluProLysValAspGluValGlnValGlyTyr                              192519301935                                                                  ValTrpAspLeuTrpGluTrpIleMetArgGlnValArgMetValMet                              194019451950                                                                  AlaArgLeuArgAlaLeuCysProValValSerLeuProLeuTrpHis                              195519601965                                                                  CysGlyGluGlyTrpSerGlyGluTrpLeuLeuAspGlyHisValGlu                              197019751980                                                                  SerArgCysLeuCysGlyCysValIleThrGlyAspValPheAsnGly                              1985199019952000                                                              GlnLeuLysGluProValTyrSerThrLysLeuCysArgHisTyrTrp                              200520102015                                                                  MetGlyThrValProValAsnMetLeuGlyTyrGlyGluThrSerPro                              202020252030                                                                  LeuLeuAlaSerAspThrProLysValValProPheGlyThrSerGly                              203520402045                                                                  TrpAlaGluValValValThrProThrHisValValIleArgArgThr                              205020552060                                                                  SerProTyrGluLeuLeuArgGlnGlnIleLeuSerAlaAlaValAla                              2065207020752080                                                              GluProTyrTyrValAspGlyIleProValSerTrpAspAlaAspAla                              208520902095                                                                  ArgAlaProAlaMetValTyrGlyProGlyGlnSerValThrIleAsp                              210021052110                                                                  GlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsnValAla                              211521202125                                                                  ProSerGluValSerSerGluValSerIleAspIleGlyThrGluThr                              213021352140                                                                  GluAspSerGluLeuThrGluAlaAspLeuProProAlaAlaAlaAla                              2145215021552160                                                              LeuGlnAlaIleGluAsnAlaAlaArgIleLeuGluProHisIleAsp                              216521702175                                                                  ValIleMetGluAspCysSerThrProSerLeuCysGlySerSerArg                              218021852190                                                                  GluMetProValTrpGlyGluAspIleProArgThrProSerProAla                              219522002205                                                                  LeuIleSerValThrGluSerSerSerAspGluLysThrProSerVal                              221022152220                                                                  SerSerSerGlnGluAspThrProSerSerAspSerPheGluValIle                              2225223022352240                                                              GlnGluSerGluThrAlaGluGlyGluGluSerValPheAsnValAla                              224522502255                                                                  LeuSerValLeuGluAlaLeuPheProGlnSerAspAlaThrArgLys                              226022652270                                                                  LeuThrValArgMetAsnCysCysValGluLysSerValThrArgPhe                              227522802285                                                                  PheSerLeuGlyLeuThrValAlaAspValAlaSerLeuCysGluMet                              229022952300                                                                  GluIleGlnAsnHisThrAlaTyrCysAspLysValArgThrProLeu                              2305231023152320                                                              GluLeuGlnValGlyCysLeuValGlyAsnGluLeuThrPheGluCys                              232523302335                                                                  AspLysCysGluAlaArgGlnGluThrLeuAlaSerPheSerTyrIle                              234023452350                                                                  TrpSerGlyValProLeuThrArgAlaThrProAlaLysProProVal                              235523602365                                                                  ValArgProValGlySerLeuLeuValAlaAspThrThrLysValTyr                              237023752380                                                                  ValThrAsnProAspAsnValGlyArgArgValAspLysValThrPhe                              2385239023952400                                                              TrpArgAlaProArgValHisAspLysTyrLeuValAspSerIleGlu                              240524102415                                                                  ArgAlaArgArgAlaAlaGlnAlaCysGlnSerMetGlyTyrThrTyr                              242024252430                                                                  GluGluAlaIleArgThrValArgProHisAlaAlaMetGlyTrpGly                              243524402445                                                                  SerLysValSerValLysAspLeuAlaThrProAlaGlyLysMetAla                              245024552460                                                                  ValHisAspArgLeuGlnGluIleLeuGluGlyThrProValProPhe                              2465247024752480                                                              ThrLeuThrValLysLysGluValPhePheLysAspArgLysGluGlu                              248524902495                                                                  LysAlaProArgLeuIleValPheProProLeuAspPheArgIleAla                              250025052510                                                                  GluLysLeuIleLeuGlyAspProGlyArgValAlaLysAlaValLeu                              251525202525                                                                  GlyGlyAlaTyrAlaPheGlnTyrThrProAsnGlnArgValLysGlu                              253025352540                                                                  MetLeuLysLeuTrpGluSerLysLysThrProCysAlaIleCysVal                              2545255025552560                                                              AspAlaThrCysPheAspSerSerIleThrGluGluAspValAlaLeu                              256525702575                                                                  GluThrGluLeuTyrAlaLeuAlaSerAspHisProGluTrpValArg                              258025852590                                                                  AlaLeuGlyLysTyrTyrAlaSerGlyThrMetValThrProGluGly                              259526002605                                                                  ValProValGlyGluArgTyrCysArgSerSerGlyValLeuThrThr                              261026152620                                                                  SerAlaSerAsnCysLeuThrCysTyrIleLysValLysAlaAlaCys                              2625263026352640                                                              GluArgValGlyLeuLysAsnValSerLeuLeuIleAlaGlyAspAsp                              264526502655                                                                  CysLeuIleIleCysGluArgProValCysAspProCysAspAlaLeu                              266026652670                                                                  GlyArgAlaLeuAlaSerTyrGlyTyrAlaCysGluProSerTyrHis                              267526802685                                                                  AlaSerLeuAspThrAlaProPheCysSerThrTrpLeuAlaGluCys                              269026952700                                                                  AsnAlaAspGlyLysArgHisPhePheLeuThrThrAspPheArgArg                              2705271027152720                                                              ProLeuAlaArgMetSerSerGluTyrSerAspProMetAlaSerAla                              272527302735                                                                  IleGlyTyrIleLeuLeuTyrProTrpHisProIleThrArgTrpVal                              274027452750                                                                  IleIleProHisValLeuThrCysAlaPheArgGlyGlyGlyThrPro                              275527602765                                                                  SerAspProValTrpCysGlnValHisGlyAsnTyrTyrLysPhePro                              277027752780                                                                  LeuAspLysLeuProAsnIleIleValAlaLeuHisGlyProAlaAla                              2785279027952800                                                              LeuArgValThrAlaAspThrThrLysThrLysMetGluAlaGlyLys                              280528102815                                                                  ValLeuSerAspLeuLysLeuProGlyLeuAlaValHisArgLysLys                              282028252830                                                                  AlaGlyAlaLeuArgThrArgMetLeuArgSerArgGlyTrpAlaGlu                              283528402845                                                                  LeuAlaArgGlyLeuLeuTrpHisProGlyLeuArgLeuProProPro                              285028552860                                                                  GluIleAlaGlyIleProGlyGlyPheProLeuSerProProTyrMet                              2865287028752880                                                              GlyValValHisGlnLeuAspPheThrSerGlnArgSerArgTrpArg                              288528902895                                                                  TrpLeuGlyPheLeuAlaLeuLeuIleValAlaLeuPheGly                                    290029052910                                                                  (2) INFORMATION FOR SEQ ID NO:184:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GV5446IRT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:                                     CGGTCCCTCGAACTCCAGCGAGTCTTTTTTTTTTTTTTT39                                     (2) INFORMATION FOR SEQ ID NO:185:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: GE-CAP from T55806                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:185:                                     MetSerLeuLeuThrAsnArgPheIleArgArgValAspLysAspGln                              151015                                                                        TrpGlyProGlyValThrGlyThrAspProGluProCysProSerArg                              202530                                                                        TrpAlaGlyLysCysMetGlyProProSerSerAlaAlaAlaCysSer                              354045                                                                        ArgGlySerProArgIleLeuArgValArgAlaGlyGlyIleSerLeu                              505560                                                                        PheTyrThrIleMetAla                                                            6570                                                                          (2) INFORMATION FOR SEQ ID NO:186:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-S59 Variant                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTTACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAG300               GTTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGGGAAGGACCTCAAG360               CCCTGCCCTTCCCGGTGGGGCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:187:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-S368 Variant                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187:                                     AGACGCAATGACTCGGCGCCAACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTTACCCACC240               CGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGAGAGGGACTCCAAG360               TCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:188:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-S309 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCCTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCATGCGGCGAGAACGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTTACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GTTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGTCACGGGGAAGGACCCCGG360               ATCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:189:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-FZ VARIANT                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:189:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGCTACCCACC240               TGGGCAAACGACGCCCATGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GATTCGTCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCCTGGGGAAGGACCCCAG360               ACCCTGCCCTTCCCGGTGGGACGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:190:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G21 VARIANT                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:190:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGTCC180               TACCGGTGTGAATAAGGACCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTTACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGGGAAGGACCCCAAG360               CCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:191:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G23 VARIANT                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:191:                                     AGACGCAATGACTCGGCGCCAACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCGCAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACATCAGGCATGTCGTTAAACCGAGCCCGTTACCCGCC240               TGGGCTAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GTTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGTTACGGGGAAGGACCCCGA360               ACCCTGCCCTTCCCGGCGGACCGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:192:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 405 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G59 VARIANT                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGGGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACTGAGCCCGTAACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAG300               GGATTATTCCCGGCGAGTTGGCAAGGACCAGTGGGGGCCGGGAGCTACAGAGAAGGACTC360               TGAGCTCTGCCCTTCCCGGTGGAACGGGAAATGCATGGGGCCACC405                              (2) INFORMATION FOR SEQ ID NO:193:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-E36 VARIANT                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:193:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGCCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCACTACCCACC240               TGGGCAAACGACGCCCACGTACGGTCTACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTAAGCCGGCGAGTTGACAAAGACCAGTGGGGGCCGGGGGTCACAGGGATGGACCCTGG360               ACCCTGCCCTTCCCGGTGGAGTGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:194:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-R38730 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGGATCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTATCCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GTTCGTCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGTTGCGGGGAAGGACCCCGA360               ACTCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:195:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G281 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:195:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGTCC180               TACCGGTGTGAATAAGGACCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTTACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGGGAAGGACCCCAAG360               CCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:196:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G157 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:196:                                     AGACGCAATGACTCGGCGCCGACCCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGGG120               AAGGTTAAGATTCCTCTTGTGCCTGTGGCGAGACAGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGACCGACACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTTGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGTGCTGGGGGAAGGACCCCCTT360               GCACCGCCCTTCCCGGTGGGACGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:197:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G154 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:197:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCCTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTACGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGCTGGCCT180               TACCGGTGTGAATAAAGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAGTAG300               GTTTAACCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGCCTTGGAGATGGACTCCAAG360               TCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:198:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G213 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:198:                                     AGACGCAATGACTCGGCGCCAACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGTCC180               TACCGGTGGGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCAAACGACGCCCACGTATGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAG300               GTTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGTTCGGGGAAGGACCCCGTA360               CCCTGCCCTTCCCGGTGGAACGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:199:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G204 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:199:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTTAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCCTGGAGAGGGACTCCAGG360               TCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:200:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G191 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:200:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCCTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGGATCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGCTAAACCGAGCCCGTATCCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GTTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGAGGTTACGGGGAAGGACCCCGA360               GCCTCGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:201:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-G299 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCAAACGACGCCCACGCACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAG300               GAGTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGAGTCACGGGGATGGACCCCGG360               GCTCTGCCCTTCCCGGTGGAACGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:202:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-T56957 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:202:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCATCACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTACAATGTCTCTCTTGACCAATAG300               GCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGTCACAGGGATGGACCCTGG360               GCCCTGCCCTTCCCGGTGGGGTGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:203:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-C01698 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:203:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGAGATGGACTCCAAG360               TCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:204:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-T27034 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:204:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCATTTCCCGCC240               TGGGCTAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GTTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGAGTCACTGGGATGGACCCAGG360               GCTCTGCCCTTCCCGGCGGGGTGGGAAAAGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:205:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-E57963 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCGCAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGAGAAGGACTCCAAG360               TCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:206:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-R37166 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:206:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTAACCCGCC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GTTTAACCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGCCTTGGAGATGGACTCCAAG360               TCCTGCCCTTCCCGGCGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:207:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 404 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-B5 VARIANT                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:207:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCCTGGTAGCCACTATAGGTGGGTCTTAAGGG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCTAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTTTTGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGTTATGGGGAAGGACCCC360               AAACCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC404                               (2) INFORMATION FOR SEQ ID NO:208:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-B33 VARIANT                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:208:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCCTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTTCCCCGCC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GTTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGATCATGGGGAAGGACCCCAG360               ATCCTGCCCTTCCCGGCGGGCCGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:209:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-FH010 VARIANT                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:209:                                     AGACGCAATGACTCGGCGCCGACCCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGGG120               AAGGTTAAGATTCCTCTTGTGCCTGTGGCGAGACAGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACTGAGACCGACACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTTGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTGGGGGAAGGACCCCCAG360               TCCTGCCCTTCCCGGTGGGACGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:210:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-PNF2161 VARIANT                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:210:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGGGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTTACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCGTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTTGGAGAGGGACTCCAAG360               TCCCGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:211:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-JC VARIANT                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:211:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGGGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTAACCCGCC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCGCTCTTGACCAATAG300               GCTTAGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGTTTATGGGGAAGGACCCCAA360               ACCCTGCCCTTCCCGGCGGACCGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:212:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-7155 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212:                                     AGACGTTATGAACCGGCGCCGCCCCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGGG120               GTGGTCAAGGTCCCTCTAGCGCTTGTGGCGAGAAAGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCATTATCCTCC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTTGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGTGCCGGGGGAAGGACCCCCGG360               TACTGCCCCTCCCGGAGGAGTGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:213:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-7244 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:                                     AGACGTTAAGAACCGGCGCCGCCCCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGGG120               GTGGTCAAGGTCCCTCTGGCGCTTGTGGCGAGAAAGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCATTACCCTCC240               TGGGCAAACGACGCCCATGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTTGCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGTGGCGGGGGAAGGACCCCCGT360               CACTGCCCTTCCCGGAGGGGTGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:214:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-K27 VARIANT                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:214:                                     AGACGTTAAGTACCGGCGCCGACCCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGGG120               TTGGTCAAGGTCCCTCTGGCGCTTGTGGCGAGAAAGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCATTACCCACC240               TGGGCAAACAACGCCCACGTACGGTCCACGTCGCCCTACAATGTCTCTCTTGACCAATAG300               GCTTTGCCGGCGAGTTGACAAGGACCAGTGGGGGCTGGGCGGCGAGGGAAGGACCCTCGT360               CGCTGCCCTTCCCGGCGGGGTGGGGAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:215:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-K30 VARIANT                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:215:                                     AGACGTTAAGAACCGGCGCCTTCCCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAGTCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGGG120               AGGGTTAAGGTCCCTCTGGCGCTTGTGGCGAGAAAGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCATTACCCACC240               TGGGCAAACAACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTTTGCCGGCGAGTTGACAAGGACCAGTGGGGGCTGGGCGGTAGGGGAAGGACCCTTGC360               CGCTGCCCTTCCCGGTGGGGTGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:216:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-T55875 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGACGAGACCGCGCACGGTCCGCAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAG300               GTTTAACCGGCGAGTTGGCAAGGACCAGTGGGGGCCGGGGGCTTGGAGAGGGACTCCAAG360               TCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:217:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-T56633 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:217:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCACTACCCACC240               TGGGCTAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAG300               GCTAGTCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGAGGTCACAGGGATGGACCCTGG360               GCCTTGCCCTTCCCGGTGGAGTGGGAAAAGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:218:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 404 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-EB20 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:218:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACCT240               GGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAGG300               AGTTATCTCCGGCGAGTTGGCAAGGACCAGTGGGGGCCGGGGGTTACGGGGAAGGACCCC360               GAACCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC404                               (2) INFORMATION FOR SEQ ID NO:219:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-T55806 VARIANT                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:219:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGCC60                AGGGTTGGTAGGTCGTAAATCCCGGTCATCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACCT240               GGGCAAACGACGCTCACGTACGGTCCACGTCGCCCTTCAATGTCTCTCTTGACCAATAGG300               TTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGTTACGGGGACGGACCCCGAA360               CCCTGCCCTTCCCGGTGGGCCGGGAAATGCATGGGGCCACC401                                  (2) INFORMATION FOR SEQ ID NO:220:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-BG34 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:220:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCACAGGTGTTGGCCC180               TACCGGTGTGAATAAGGGCCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGTCACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAG300               GAGTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGAGTCACGGGGATGGACCCCGG360               GCTCTGCCCTTCCCGGTGGAACGGGAAACGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:221:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-BE12 VARIANT                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221:                                     AGACGCAATGACTCGGCGCCGACTCGGCGACCGGCCAAAAGGTGGTGGATGGGTGATGAC60                AGGGTTGGTAGGTCGTAAATCCCGGTCACCTTGGTAGCCACTATAGGTGGGTCTTAAGAG120               AAGGTTAAGATTCCTCTTGTGCCTGCGGCGAGACCGCGCACGGTCCGCAGGTGTTGGTCC180               TACCGGTGTGAATAAGGACCCGACGTCAGGCTCGTCGTTAAACCGAGCCCGCCACCCACC240               TGGGCAAACGACGCCCACGTACGGTCCACGTCGCCCTTCAATGCCTCTCTTGGCCAATAG300               GTTTATCCGGCGAGTTGACAAGGACCAGTGGGGGCCGGGGGCTCCGGGGAAGAACCCCGA360               GCCCCGCCCTTCCCGGTGGGACGGGAAATGCATGGGGCCACC402                                 (2) INFORMATION FOR SEQ ID NO:222:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-FORWARD PRIMER                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:222:                                     CCAAAAGGTGGTGGATGGGTGATG24                                                    (2) INFORMATION FOR SEQ ID NO:223:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-FORWARD PRIMER                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:223:                                     GTGATGMCAGGGTTGGTAGGTCGT24                                                    (2) INFORMATION FOR SEQ ID NO:224:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-FORWARD PRIMER                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224:                                     GGTAGCCACTATAGGTGGGTCTTAAG26                                                  (2) INFORMATION FOR SEQ ID NO:225:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-REVERSE PRIMER                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:225:                                     GAGMGRCATTGWAGGGCGACGTRGA25                                                   (2) INFORMATION FOR SEQ ID NO:226:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-REVERSE PRIMER                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226:                                     GRCATTGWAGGGCGACGTRGA21                                                       (2) INFORMATION FOR SEQ ID NO:227:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HGV-REVERSE PRIMER                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:227:                                     CCCCACTGGTCYTTGYCAACTC22                                                      (2) INFORMATION FOR SEQ ID NO:228:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PRIMER GV75- 36FE                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:228:                                     GCGAGATCTAAAATGCAGGCCTGATGGGT29                                               (2) INFORMATION FOR SEQ ID NO:229:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PRIMER GV75- 7064RLE                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:229:                                     GCGAGATCTAAAATGTGGACTGCTAAGCC29                                               (2) INFORMATION FOR SEQ ID NO:230:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PRIMER FV94- 28F                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:230:                                     GCGAGATCTAAAATGGCAAGCCCCAGAAACCGACGCCTATCTAAGT46                              (2) INFORMATION FOR SEQ ID NO:231:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PRIMER FV94- 2864R                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:231:                                     GGCATGATGAATTCGCAACGAGGGCCGGGACACCAAGAT39                                     (2) INFORMATION FOR SEQ ID NO:232:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PRIMER FV94- 6439F                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:232:                                     GCGAGATCTAAAATGGGCCTCCGACACCCCGAAGGTTGT39                                     (2) INFORMATION FOR SEQ ID NO:233:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: PRIMER FV94- 9331R                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:233:                                     GCGAGATCTGAATTCTTCCCGGGGTGCACCCCTTCAGAT39                                     (2) INFORMATION FOR SEQ ID NO:234:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9327 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 3ZHGV-6, HGV FROM PNF2161                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:234:                                     GCAAGCCCCAGAAACCGACGCCTATCTAAGTAGACGCAATGACTCGGCGCCGACTCGGCG60                ACCGGCCAAAAGGTGGTGGATGGGTGATGACAGGGTTGGTAGGTCGTAAATCCCGGTCAC120               CTTGGTAGCCACTATAGGTGGGTCTTAAGAGAAGGTTAAGATTCCTCTTGTGCCTGCGGC180               GAGACCGCGCACGGTCCACAGGTGTTGGCCCTACCGGTGGGAATAAGGGCCCGACGTCAG240               GCTCGTCGTTAAACCGAGCCCGTTACCCACCTGGGCAAACGACGCCCACGTACGGTCCAC300               GTCGCCCTTCAATGTCTCTCTTGACCAATAGGCGTAGCCGGCGAGTTGACAAGGACCAGT360               GGGGGCCGGGGGCTTGGAGAGGGACTCCAAGTCCCGCCCTTCCCGGTGGGCCGGGAAATG420               CATGGGGCCACCCAGCTCCGCGGCGGCCTGCAGCCGGGGTAGCCCAAGAATCCTTCGGGT480               GAGGGCGGGTGGCATTTCCTTTTTCTATACCATCATGGCAGTCCTTCTGCTCCTTCTCGT540               GGTTGAGGCCGGGGCCATTCTGGCCCCGGCCACCCACGCTTGTCGAGCGAATGGGCAATA600               TTTCCTCACAAATTGTTGTGCCCCGGAGGACATCGGGTTCTGCCTGGAGGGTGGATGCCT660               GGTGGCCCTGGGGTGCACGATTTGCACTGACCAATGCTGGCCACTGTATCAGGCGGGTTT720               GGCTGTGCGGCCTGGCAAGTCCGCGGCCCAACTGGTGGGGGAGCTGGGTAGCCTATACGG780               GCCCCTGTCGGTCTCGGCCTATGTGGCTGGGATCCTGGGCCTGGGTGAGGTGTACTCGGG840               TGTCCTAACGGTGGGAGTCGCGTTGACGCGCCGGATCTACCCGGTGCCTAACCTGACGTG900               TGCAGTCGCGTGTGAGTTAAAGTGGGAAAGTGAGTTTTGGAGATGGACTGAACAGCTGGC960               CTCCAACTACTGGATTCTGGAATACCTCTGGAAGGTCCCATTTGATTTCTGGAGAGGCGT1020              GATAAGCCTGACCCCCTTGTTGGTTTGCGTGGCCGCATTGCTGCTGCTTGAGCAACGGGT1080              TGTCATGGTCTTCCTGTTGGTGACGATGGCCGGGATGTCGCAAGGCGCCCCTGCCTCCGT1140              TTTGGGGTCACGCCCCTTTGACTACGGGTTGACTTGGCAGACCTGCTCTTGCAGGGCCAA1200              CGGTTCGCGTTTTTCGACTGGGGAGAAGGTGTGGGACCGTGGGAACGTTACGCTTCAGTG1260              TGACTGCCCTAACGGCCCCTGGGTGTGGTTGCCAGCCTTTTGCCAAGCAATCGGCTGGGG1320              TGACCCCATCACTTATTGGAGCCACGGGCAAAATCAGTGGCCCCTTTCATGCCCCCAGTA1380              TGTCTATGGGTCTGCTACAGTCACTTGCGTGTGGGGTTCCGCTTCTTGGTATGCCTCCAC1440              CAGTGGTCGCGACTCGAAGATAGATGTGTGGAGTTTAGTGCCAGTTGGCTCTGCCACCTG1500              CACCATAGCCGCACTTGGATCATCGGATCGCGACACGGTGCCTGGGCTCTCCGAGTGGGG1560              AATCCCGTGCGTGACGTGTGTTCTGGACCGTCGGCCTGCTTCATGCGGCACCTGTGTGAG1620              GGACTGCTGGCCCGAGACCGGGTCGGTTAGGTTCCCATTCCATCGGTGCGGCGTGGGGCC1680              TCGGCTGACAAAGGACTTGGAAGCTGTGCCCTTCGTCAATAGGACAACTCCCTTCACCAT1740              TAGGGGGCCCCTGGGCAACCAGGGCCGAGGCAACCCGGTGCGGTCGCCCTTGGGTTTTGG1800              GTCCTACGCCATGACCAGGATCCGAGATACCCTACATCTGGTGGAGTGTCCCACACCAGC1860              CATCGAGCCTCCCACCGGGACGTTTGGGTTCTTCCCCGGGACGCCGCCTCTCAACAACTG1920              CATGCTCTTGGGCACGGAAGTGTCCGAGGCACTTGGGGGGGCTGGCCTCACGGGGGGGTT1980              CTATGAACCCCTGGTGCGCAGGTGTTCGGAGCTGATGGGAAGCCGAAATCCGGTTTGTCC2040              GGGGTTTGCATGGCTCTCTTCGGGCAGGCCTGATGGGTTTATACATGTCCAGGGTCACTT2100              GCAGGAGGTGGATGCAGGCAACTTCATCCCGCCCCCGCGCTGGTTGCTCTTGGACTTTGT2160              ATTTGTCCTGTTATACCTGATGAAGCTGGCTGAGGCACGGTTGGTCCCGCTGATCTTGCT2220              GCTGCTATGGTGGTGGGTGAACCAGCTGGCAGTCCTAGGGCTGCCGGCTGTGGAAGCCGC2280              CGTGGCAGGTGAGGTCTTCGCGGGCCCTGCCCTGTCCTGGTGTCTGGGACTCCCGGTCGT2340              CAGTATGATATTGGGTTTGGCAAACCTGGTGCTGTACTTTAGATGGTTGGGACCCCAACG2400              CCTGATGTTCCTCGTGTTGTGGAAGCTTGCTCGGGGAGCTTTCCCGCTGGCCCTCTTGAT2460              GGGGATTTCGGCGACCCGCGGGCGCACCTCAGTGCTCGGGGCCGAGTTCTGCTTCGATGC2520              TACATTCGAGGTGGACACTTCGGTGTTGGGCTGGGTGGTGGCCAATGTGGTAGCTTGGGC2580              CATTGCGCTCCTGAGCTCGATGAGCGCAGGGGGGTGGAGGCACAAAGCCGTGATCTATAG2640              GACGTGGTGTAAGGGGTACCAGGCAATCCGTCAAAGGGTGGTGAGGAGCCCCCTCGGGGA2700              GGGGCGGCCTGCCAAACCCCTGACCTTTGCCTGGTGCTTGGCCTCGTACATCTGGCCAGA2760              TGCTGTGATGATGGTGGTGGTTGCCTTGGTTCTTCTCTTTGGCCTGTTCGACGCGTTGGA2820              TTGGGCCTTGGAGGAGATCTTGGTGTCCCGGCCCTCGCTGCGGCGTTTGGCTCGGGTGGT2880              TGAGTGCTGTGTGATGGCGGGTGAGAAGGCCACAACCGTCCGGCTGGTCTCCAAGATGTG2940              TGCGAGAGGAGCTTATTTGTTCGATCATATGGGCTCATTTTCGCGTGCTGTCAAGGAGCG3000              CCTGTTGGAATGGGACGCGGCTCTTGAACCTCTGTCATTCACTAGGACGGACTGTCGCAT3060              CATACGGGATGCCGCGAGGACTTTGTCCTGCGGGCAATGCGTCATGGGTTTACCCGTGGT3120              TGCGCGCCGTGGTGATGAGGTTCTCATCGGCGTCTTCCAGGATGTGAATCATTTGCCTCC3180              CGGGTTTGTTCCGACCGCGCCTGTTGTCATCCGACGGTGCGGAAAGGGCTTCTTGGGGGT3240              CACAAAGGCTGCCTTGACAGGTCGGGATCCTGACTTACATCCAGGGAACGTCATGGTGTT3300              GGGGACGGCTACGTCGCGAAGCATGGGAACATGCTTGAACGGCCTGCTGTTCACGACCTT3360              CCATGGGGCTTCATCCCGAACCATCGCCACACCCGTGGGGGCCCTTAATCCCAGATGGTG3420              GTCAGCCAGTGATGATGTCACGGTGTATCCACTCCCGGATGGGGCTACTTCGTTAACGCC3480              TTGTACTTGCCAGGCTGAGTCCTGTTGGGTCATCAGATCCGACGGGGCCCTATGCCATGG3540              CTTGAGCAAGGGGGACAAGGTGGAGCTGGATGTGGCCATGGAGGTCCCTGATTTCCGTGG3600              CTCGTCTGGCTCACCGGTCCTATGTGACGAGGGGCACGCAGTAGGAATGCTCGTGTCTGT3660              GCTTCACTCCGGTGGTAGGGTCACCGCGGCACGGTTCACTAGGCCGTGGACCCAAGTGCC3720              AACAGATGCCAAAACCACCACTGAACCCCCTCCGGTGCCGGCCAAAGGAGTTTTCAAAGA3780              GGCCCCGTTGTTTATGCCTACGGGAGCGGGAAAGAGCACTCGCGTCCCGTTGGAGTACGG3840              CAACATGGGGCACAAGGTCTTAGTCTTGAACCCCTCAGTGGCCACTGTGCGGGCCATGGG3900              CCCGTACATGGAGCGGCTGGCGGGTAAACATCCAAGTATATACTGTGGGCATGATACAAC3960              TGCTTTCACAAGGATCACTGACTCCCCCCTGACGTATTCAACCTATGGGAGGTTTTTGGC4020              CAACCCTAGGCAGATGCTACGGGGCGTTTCGGTGGTCATTTGTGATGAGTGCCACAGTTA4080              TGACTCAACCGTGCTGTTAGGCATTGGGAGGGTTCGGGAGCTGGCGCGTGGGTGCGGAGT4140              GCAACTAGTGCTCTACGCCACCGCTACGCCTCCCGGATCCCCTATGACGCAGCACCCTTC4200              CATAATTGAGACAAAATTGGACGTGGGCGAGATTCCCTTTTATGGGCACGGAATACCCCT4260              CGAGCGGATGCGAACCGGAAGGCACCTCGTGTTCTGCCATTCTAAGGCTGAGTGCGAGCG4320              CCTTGCTGGCCAGTTCTCCGCTAGGGGGGTCAATGCCATTGCCTATTATAGGGGTAAAGA4380              CAGTTCTATCATCAAGGATGGGGACCTGGTGGTCTGTGCCACAGACGCGCTTTCCACTGG4440              GTACACTGGAAATTTCGACTCCGTCACCGACTGTGGATTAGTGGTGGAGGAGGTCGTTGA4500              GGTGACCCTTGATCCTACCATTACCATCTCCCTGCGGACAGTGCCTGCGTCGGCTGAACT4560              GTCGATGCAAAGACGAGGACGCACGGGTAGGGGCAGGTCTGGACGCTACTACTACGCGGG4620              GGTGGGCAAAGCCCCTGCGGGTGTGGTGCGCTCAGGTCCTGTCTGGTCGGCGGTGGAAGC4680              TGGAGTGACCTGGTACGGAATGGAACCTGACTTGACAGCTAACCTACTGAGACTTTACGA4740              CGACTGCCCTTACACCGCAGCCGTCGCGGCTGATATCGGAGAAGCCGCGGTGTTCTTCTC4800              TGGGCTCGCCCCATTGAGGATGCACCCTGATGTCAGCTGGGCAAAAGTTCGCGGCGTCAA4860              CTGGCCCCTCTTGGTGGGTGTTCAGCGGACCATGTGTCGGGAAACACTGTCTCCCGGCCC4920              ATCGGATGACCCCCAATGGGCAGGTCTGAAGGGCCCAAATCCTGTCCCACTCCTGCTGAG4980              GTGGGGCAATGATTTACCATCTAAAGTGGCCGGCCACCACATAGTGGACGACCTGGTCCG5040              GAGACTCGGTGTGGCGGAGGGTTACGCCCGCTGCGACGCTGGGCCGATCTTGATGATCGG5100              TCTAGCTATCGCGGGGGGAATGATCTACGCGTCGTACACCGGGTCGCTAGTGGTGGTGAC5160              AGACTGGGATGTGAAGGGGGGTGGCGCCCCCCTTTATCGGCATGGAGACCAGGCCACGCC5220              TCAGCCGGTGGTGCAGGTTCCTCCGGTAGACCATCGGCCGGGGGGTGAATCAGCACCATC5280              GGATGCCAAGACAGTGACAGATGCGGTGGCAGCGATCCAGGTGGACTGCGATTGGACTAT5340              CATGACTCTGTCGATCGGAGAAGTGTTGTCCTTGGCTCAGGCTAAGACGGCCGAGGCCTA5400              CACAGCAGCCACCAAGTGGCTCGCTGGCTGCTATACGGGGACGCGGGCCGTTCCCACTGT5460              ATCCATTGTTGACAAGCTCTTCGCCGGAGGGTGGGCGGCTGTGGTGGGCCATTGCCACAA5520              CGTGATTGCTGCGGCGGTGGCGGCCTACGGGGCTTCAAAGAGCCCGCCGTTGGCAGCCGC5580              GGCTTCCTACCTGATGGGGTTGGGCGTTGGAGGCAACGCTCAGACGCGTCTGGCATCTGC5640              CCTCCTATTGGGGGCTGCTGGAACCGCCTTGGGCACTCCTGTCGTGGGCTTGACCATGGC5700              AGGTGCGTTCATGGGGGGCGCCAGTGTCTCCCCCTCCTTGGTCACCATTTTATTGGGGGC5760              CGTCGGAGGTTGGGAGGGTGTTGTCAACGCGGCGAGCCTAGTCTTTGACTTCATGGCGGG5820              GAAACTTTCATCAGAAGATCTGTGGTATGCCATCCCGGTACTGACCAGCCCGGGGGCGGG5880              CCTTGCGGGGATCGCTCTCGGGTTGGTTTTGTATTCAGCTAACAACTCTGGCACTACCAC5940              TTGGTTGAACCGTCTGCTGACTACGTTACCAAGGTCTTCATGTATCCCGGACAGTTACTT6000              TCAGCAAGTTGACTATTGCGACAAGGTCTCAGCCGTGCTCCGGCGCCTGAGCCTCACCCG6060              CACAGTGGTTGCCCTGGTCAACAGGGAGCCTAAGGTGGATGAGGTACAGGTGGGGTATGT6120              CTGGGACCTGTGGGAGTGGATCATGCGCCAAGTGCGCGTGGTCATGGCCAGACTCAGGGC6180              CCTCTGCCCCGTGGTGTCATTACCCTTGTGGCACTGCGGGGAGGGGTGGTCCGGGGAATG6240              GTTGCTTGACGGTCATGTTGAGAGTCGCTGCCTCTGTGGCTGCGCGATCACTGGTGACGT6300              TCTGAATGGGCAACTCAAAGAACCAGTTTACTCTACCAAGCTGTGCCGGCACTATTGGAT6360              GGGGACTGTCCCTGTGAACATGCTGGGTTACGGTGAAACGTCGCCTCTCCTGGCCTCCGA6420              CACCCCGAAGGTTGTGCCCTTCGGGACGTCTGGCTGGGCTGAGGTGGTGGTGACCACTAC6480              CCACGTGGTAATCAGGAGAACCTCCGCCTATAAGCTGCTGCGCCAGCAAATCCTATCGGC6540              TGCTGTAGCTGAGCCCTACTACGTCGACGGCATTCCGGTCTCATGGGACGCGGACGCTCG6600              TGCGCCCGCCATGGTCTATGGCCCTGGGCAAAGTGTTACCATTGACGGGGAGCGCTACAC6660              CCTGCCTCATCAACTGAGGCTCAGGAATGTGGCGCCCTCTGAGGTTTCATCCGAGGTGTC6720              CATTGACATTGGGACGGAGACTGGAGACTCAGAACTGACTGAGGCCGATCTGCCGCCGGC6780              GGCTGCTGCTCTCCAAGCGATCGAGAATGCTGCGAGGATTCTTGAACCGCACATTGATGC6840              CATCATGGAGGACTGCAGTACACCCTCTCTTTGTGGTAGTAGCCGAGAGATGCCTGTATG6900              GGGAGAAGACATCCCCCGTACTCCATCGCCAGCACTTATCTCGGTTACTGAGAGCAGCTC6960              AGATGAGAAGACCCCGTCGGTGTCCTCCTCGCAGGAGGATACCCCGTCCTCTGACTCATT7020              CGAGGTCATCCAAGAGTCCGAGACAGCCGAAGGGGAGGAAAGCGTCTTCAACGTGGCTCT7080              TTCCGTATTAGAAGCCTCATTTCCACAGAGCGACGCGACCAGGAAGCTTACCGTCAAGAT7140              GTCGTGCTGCGTTGAAAAGAGCGTCACGCGCTTTTTCTCATTGGGGTTGACGGTGGCTGA7200              TGTTGCTAGCCTGTGTGAGATGGAAATCCAGAACCATACAGCCTATTGTGACAAGGTGCG7260              CACTCCGCTTGAATTGCAGGTTGGGTGCTTGGTGGGCAATGAACTTACCTTTGAATGTGA7320              CAAGTGTGAGGCTAGGCAAGAAACCTTGGCCTCCTTCTCTTACATTTGGTCTGGAGTGCC7380              GCTGACTAGGGCCACGCCGGCCAAGCCTCCCGTGGTGAGGCCGGTTGGCTCTTTATTAGT7440              GGCCGACACTACTAAGGTGTATGTTACCAATCCAGACAATGTGGGACGGAGGGTGGACAA7500              GGTGACCTTCTGGCGTGCTCCTAGGGTTCATGATAAGTACCTCGTGGACTCTATTGAGCG7560              CGCTAAGAGGGCCGCTCAAGCCTGCCTAAGCATGGGTTACACTTATGAGGAAGCAATAAG7620              GACTGTAAGGCCACATGCTGCCATGGGCTGGGGATCTAAGGTGTCGGTTAAGGACTTAGC7680              CACCCCCGCGGGGAAGATGGCCGTCCATGACCGGCTCCAGGAGATACTTGAAGGGACTCC7740              GGTCCCCTTTACTCTTACTGTGAAAAAGGAGGTGTTCTTCAAAGACCGGAAGGAGGAGGA7800              GGCCCCCCGCCTCATTGTGTTCCCCCCCCTGGACTTCCGGATAGCTGAAAAGCTCATCTT7860              GGGAGACCCAGACCGGGTAGCCAAGGCGGTGTTGGGGGGGGCCTACGCCTTCCAGTACAC7920              CCCAAATCAGCGAGTTAAGGAGATGCTCAAGCTATGGGAGTCTAAGAAGACCCCTTGCGC7980              CATCTGTGTGGACGCCACCTGCTTCGACAGTAGCATAACTGAAGAGGACGTGGCTTTGGA8040              GACAGAGCTGTACGCTCTGGCCTCTGACCATCCAGAATGGGTGCGGGCACTTGGGAAATA8100              CTATGCCTCAGGCACCATGGTCACCCCGGAAGGGGTGCCCGTCGGTGAGAGGTATTGCAG8160              ATCCTCGGGTGTCCTAACAACTAGCGCGAGCAACTGCTTGACCTGCTACATCAAGGTGAA8220              AGCCGCCTGTGAGAGGGTGGGGCTGAAGAATGTCTCTCTTCTCATAGCCGGCGATGACTG8280              CTTGATCATATGTGAGCGGCCAGTGTGCGACCCAAGCGACGCTTTGGGCAGAGCCCTAGC8340              GAGCTATGGGTACGCGTGCGAGCCCTCATATCATGCATCCTTGGACACGGCCCCCTTCTG8400              CTCCACTTGGCTTGCTGAGTGCAATGCAGATGGGAAGCGCCATTTCTTCCTGACCACGGA8460              CTTCCGGAGGCCGCTCGCTCGCATGTCGAGTGAGTATAGTGACCCGATGGCTTCGGCGAT8520              CGGTTACATCCTCCTTTATCCTTGGCACCCCATCACACGGTGGGTCATCATCCCTCATGT8580              GCTAACGTGCGCATTCAGGGGTGGAGGCACACCGTCTGATCCGGTTTGGTGCCAGGTACA8640              TGGTAACTACTACAAGTTTCCACTGGACAAACTGCCTAACATCATCGTGGCCCTCCACGG8700              ACCAGCAGCGTTGAGGGTTACCGCAGACACAACTAAAACAAAGATGGAGGCTGGTAAGGT8760              TCTGAGCGACCTCAAGCTCCCTGGCTTAGCAGTCCACCGAAAGAAGGCCGGGGCGTTGCG8820              AACACGCATGCTCCGCTCGCGCGGTTGGGCTGAGTTGGCTAGGGGCTTGTTGTGGCATCC8880              AGGCCTACGGCTTCCTCCCCCTGAGATTGCTGGTATCCCGGGGGGTTTCCCTCTCTCCCC8940              CCCCTATATGGGGGTGGTACACCAATTGGATTTTACAAGCCAGAGGAGTCGCTGGCGGTG9000              GTTGGGGTTCTTAGCCCTGCTCATCGTAGCCCTCTTCGGGTGAACTAAATTCATCTGTTG9060              CGGCGAGGTCTGGTGACTGATCGTCACCGGAGGAGGTTCCCGCCCTCCCCGCCCCAGGGG9120              TCTCCCCGCTGGGTAAAAAGGGCCCGGCCTTGGGAGGCATGGTGGTTACTAACCCCCTGG9180              CAGGGTTAAAGCCTGATGGTGCTAATGCACTGCCACTTCGGTGGCGGGTCGCTACCTTAT9240              AGCGTAATCCGTGACTACGGGCTGCTCGCAGAGCCCTCCCCGGATGGGGCACAGTGCACT9300              GAGATCTGAAGGGGTGCACCCCGGGAA9327                                               (2) INFORMATION FOR SEQ ID NO:235:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GLI- F                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:235:                                     TAGCATGGCCTTTGCAGGGCTG22                                                      (2) INFORMATION FOR SEQ ID NO:236:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GLI- R                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:236:                                     AAGCTGTGACCGTCTCCG18                                                          (2) INFORMATION FOR SEQ ID NO:237:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE1- NF                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:237:                                     GCCGCCATGGCGGGGAAACTTTCATCAGAAG31                                             (2) INFORMATION FOR SEQ ID NO:238:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE1- NR                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:238:                                     GCGCGGATCCTAGTGACACCACGGGGCAGAGG32                                            (2) INFORMATION FOR SEQ ID NO:239:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE57F                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:239:                                     GCCGCCATGGCTCTCTTGACCAATAGGTTTATC33                                           (2) INFORMATION FOR SEQ ID NO:240:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GE57R                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:240:                                     GCGCGGATCCAGAAATGCCACCCGCCCTCAC31                                             (2) INFORMATION FOR SEQ ID NO:241:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: GE57 amino acid sequence                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:241:                                     MetSerLeuLeuThrAsnArgPheIleArgArgValAspLysAspGln                              151015                                                                        TrpGlyProGlyValThrGlyThrAspProGluProCysProSerArg                              202530                                                                        TrpAlaGlyLysCysMetGlyProProSerSerAlaAlaAlaCysSer                              354045                                                                        ArgGlySerProArgIleLeuArgValArgAlaGlyGly                                       505560                                                                        (2) INFORMATION FOR SEQ ID NO:242:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer for E1                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:242:                                     GCGCAGATCTAAAATGAGCCGTGGTGGCATTTCCTTTTTCTATACCATCATG52                        (2) INFORMATION FOR SEQ ID NO:243:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer for E1                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:243:                                     GCGCAGATCTCCAGAAATCAAATGGGACCTTCCAGAGG38                                      (2) INFORMATION FOR SEQ ID NO:244:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer for E2 with insect                     signal sequence                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:244:                                     CGCGAGATCTGTCGCAAGGCGCCCCT26                                                  (2) INFORMATION FOR SEQ ID NO:245:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer for E2 with insect                     signal sequence                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:245:                                     GCGCAGATCTAGTTGCCTGCATCCACCT28                                                (2) INFORMATION FOR SEQ ID NO:246:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer for E2 with HGV                        signal sequence                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:246:                                     CGCGAGATCTAAAATGAAACTGCTTGTCATGGTCTTCCTGTT42                                  (2) INFORMATION FOR SEQ ID NO:247:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer for E2 with HGV                        signal sequence                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:247:                                     GCGCAGATCTAGTTGCCTGCATCCACCT28                                                (2) INFORMATION FOR SEQ ID NO:248:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer for NS2a                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:248:                                     GCGCAGATCTGGCCGTGGCAGGTGAGGTCTTCGC34                                          (2) INFORMATION FOR SEQ ID NO:249:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer for NS2a                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:249:                                     GCGCAGATCTTAACGCCGCAACGAGGGCCGG31                                             (2) INFORMATION FOR SEQ ID NO:250:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer for NS2b                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:250:                                     GCGCGGATCCAAAATGATCGCTCGGGTGGTTGAGTGCTGTGTGATG46                              (2) INFORMATION FOR SEQ ID NO:251:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer for NS2b                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:251:                                     GCGCGGATCCAGGCGCGGTCGGAACAAACCCG32                                            (2) INFORMATION FOR SEQ ID NO:252:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer NS3                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:252:                                     GCGAGATCTAAAATGTGCGGAAAGGGCTTCTTGGGGGTC39                                     (2) INFORMATION FOR SEQ ID NO:253:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer NS3                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:253:                                     GCGAGATCTCATCTCCGGACCAGGTCGTCCACTATGTGG39                                     (2) INFORMATION FOR SEQ ID NO:254:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer NS4a                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:254:                                     GGCGGATCCAAAATGATCGGTGTGGCGGAGG31                                             (2) INFORMATION FOR SEQ ID NO:255:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer NS4a                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:255:                                     GGCGGGATCCATGCGCCGGAGCACGG26                                                  (2) INFORMATION FOR SEQ ID NO:256:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer NS4b                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:256:                                     GCGGGATCCAAAATGATCAGCCTCACCCGCACAG34                                          (2) INFORMATION FOR SEQ ID NO:257:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer NS5a                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:257:                                     GGCGGGATCCTACCTCCTGATTACCACGT29                                               (2) INFORMATION FOR SEQ ID NO:258:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer NS5a                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:258:                                     GCGAGATCTAAAATGACCTCCGCCTATAAGCTGCTGCGCCAG42                                  (2) INFORMATION FOR SEQ ID NO:259:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer NS5a                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:259:                                     GGCAGATCTACCTCCGTCCCACATTGTCTGGATTGGTAAC40                                    (2) INFORMATION FOR SEQ ID NO:260:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer NS5b                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:260:                                     GCGAGATCTAAAATGGTGGACAAGGTGACCTTCTGGCGTGCTC43                                 (2) INFORMATION FOR SEQ ID NO:261:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer NS5b                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:261:                                     GCGAGATCTCACCCGAAGAGGGCTACGATGAGCAGG36                                        (2) INFORMATION FOR SEQ ID NO:262:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Forward Primer E1-E2-NS2a                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:262:                                     GCGCAGATCTAAAATGAGCCGTGGTGGCATTTCCTTTTTCTATACCATCATG52                        (2) INFORMATION FOR SEQ ID NO:263:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Reverse Primer E1-E2-NS2a                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:263:                                     GCGCAGATCTTAACGCCGCAACGAGGGCCGG31                                             (2) INFORMATION FOR SEQ ID NO:264:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 9E3- REV                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:264:                                     GCTGGCTGAGGCACGGTTGGTC22                                                      (2) INFORMATION FOR SEQ ID NO:265:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer E39- 94PR                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:265:                                     CACCATCATCACAGCATCTGGC22                                                      (2) INFORMATION FOR SEQ ID NO:266:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- F12                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:266:                                     GCAACCATGGAACCTGCCAAACCCCTGACCTT32                                            (2) INFORMATION FOR SEQ ID NO:267:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- R12                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:267:                                     AGCCCCATGGAAGGTCGTGAA21                                                       (2) INFORMATION FOR SEQ ID NO:268:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- F14                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:268:                                     TTGGGATCCCTCGTGTTCCGCCATTCTAAG30                                              (2) INFORMATION FOR SEQ ID NO:269:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- R13                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:269:                                     TATGGATCCTGGTAAATCATTGCCCCACCT30                                              (2) INFORMATION FOR SEQ ID NO:270:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer 470EP- F8                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:270:                                     GCTGAATTCGCCATGGCGACGTGCGCATTCAGGGGTGGA39                                     (2) INFORMATION FOR SEQ ID NO:271:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Primer GEP- R14                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:271:                                     GGAGGATCCGCGACCCGCCACCGAAGT27                                                 (2) INFORMATION FOR SEQ ID NO:272:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Y5 epitope                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:272:                                     IleAspGlyGluArgTyrThrLeuProHisGlnLeuArgLeuArgAsn                              151015                                                                        ValAlaProSerGluValSerSerGluValSerIleAspIleGlyThr                              202530                                                                        GluAlaGluAsnSerGluLeuThrGluAlaAspLeuProProAlaAla                              354045                                                                        (2) INFORMATION FOR SEQ ID NO:273:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Q9 Epitope                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:273:                                     CysGlyLeuLeuThrArgHisHisThrAlaLeuAsnHisProSerGln                              151015                                                                        ThrProGlnArgGlyProGlyHisGlnAspLeuLeuGlnGlyProIle                              202530                                                                        GlnArgValGluGlnAlaLysGluLysAspGlnGlyAsnHisHisHis                              354045                                                                        HisHisSerIleTrpProAsp                                                         5055                                                                          (2) INFORMATION FOR SEQ ID NO:274:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Q11 Epitope                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:274:                                     AlaAlaValAlaGluProTyrTyrValAspGlyIleProValSerTrp                              151015                                                                        AspAlaAspAlaArgAlaProAlaMetValTyrGlyProGlyGlnSer                              202530                                                                        ValThrIle                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:275:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 225 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Q7-12-1 env clone                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:275:                                     GTGCCCTTCGTCAACAGGACAACTCTCTTCACCATTAGGGGGCCCCTGGGCAACCAGGGC60                CGAGGCAACCCGGTGCGGTCGCCCTTGGGTTTTGGGTCCTACGCCATGACCAGGATCCGA120               GATACCCTACATCTGGTGGAGTGTCCCACACCAGCCATCGAGCCTCCCACCGGGACGTCT180               GGGTTCTTCCCCGGGACGCCGCCTCTCAACAACTGCATGCATATG225                              (2) INFORMATION FOR SEQ ID NO:276:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 192 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Y12-15-1 NS3 clone DNA                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:276:                                     AACATGGGGCACAAGGTCTTAATCTTGAACCCCTCAGTGGCCACTGTGCGGGCCATGGGC60                CCGTACATGGAGCGGCTGGCGGGTAAACATCCAAGTATATACTGTGGGCATGATACAACT120               GCTTTCACAAGGATCACTGACTCCCCCCTGACGTATTCAACCTATGGGAGGTTTTTGGCC180               AACCCTAGGCAA192                                                               (2) INFORMATION FOR SEQ ID NO:277:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 264 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Y12-10-2 NS3 clone                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:277:                                     CCCCTCGAGCGGATGCGAACCGGAAGGCACCTCGTGTTCTGCCATTCTAAGGCTGAGTGC60                GAGCGCCTTGCTGGCCAGTTCTCCGCTAGGGGGGTCAATGCCATTGCCTATTATAGGGGT120               AAAGACAGCTCTATCATCAAGGATGGGGACCTGGTGGTCTGTGCTACAGACGCGCTTTCC180               ACTGGGTACACTGGAAATTTCGACTCCGTCACCGACTGTGGATTAGTGGTGGAGGAGGTC240               GTTGAGGTGACCCTTGATCCCACC264                                                   __________________________________________________________________________

It is claimed:
 1. A purified antibody preparation made against apolypeptide of SEQ ID NO: 15 or a fragment thereof which is specificallyimmunoreactive with Non-A Non-B Non-C Non-D Non-E Hepatitis G Virus(HGV), where HGV is characterized by the following: (i) elevated serumalanine aminotransferase levels in an infected primate, (ii) serologicdistinctness from hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus(HEV), and (iii) a viral genome comprising a polynucleotide region thatis selectively hybridizable with SEQ ID NO:19.
 2. The preparation ofclaim 1 comprising an anti-Non-A Non-B Non-C Non-D Non-E Hepatitis GVirus (HGV) polyclonal antibody.
 3. The preparation of polyclonalantibodies of claim 2, where said polyclonal antibodies are obtained byaffinity chromatography.
 4. A method for producing an antibody to Non-ANon-B Non-C Non-D Non-E Hepatitis G Virus (HGV), comprisingadministeringto a test subject a polypeptide of SEQ ID NO: 15 or a fragment thereofwhich is specifically immunoreactive with antibodies directed againstNon-A Non-B Non-C Non-D Non-E Hepatitis G Virus (HGV) in an amountsufficient to produce an immune response, wherein HGV is characterizedby the following: (i) elevated serum alanine aminotransferase levels inan infected primate, (ii) serologic distinctness from hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis Dvirus (HDV) and hepatitis E virus (HEV), and (iii) a viral genomecomprising a polynucleotide region that is selectively hybridizable withSEQ ID NO: 19, and obtaining from the subject, said antibody.
 5. Adiagnostic kit for use in screening a biological fluid sample containinga Non-A Non-B Non-C Non-D Non-E Hepatitis G Virus (HGV) polypeptideantigen, comprisingthe anti-HGV antibody preparation of claim 1, and areporter for detecting the binding of said polypeptide antigen to saidantibody.
 6. The kit of claim 5, where said antibody is a monoclonalantibody.
 7. The kit of claim 5, where said antibody is attached to asolid support.
 8. The kit of claim 5, wherein said reporter comprises asecond antibody preparation consisting of a labeled monoclonal antibody.9. The kit of claim 5, where said reporter comprises a labeled,competing antigen.
 10. The method of detecting Non-A Non-B Non-C Non-DNon-E Hepatitis G Virus (HGV) in a test subject, comprisingreacting abiological fluid sample from a test subject with an antibody of the kitof claim 5, and examining the antibody for the presence of boundantigen.
 11. The preparation of claim 1 comprising an anti- Non-A Non-BNon-C Non-D Non-E Hepatitis G Virus (HGV) monoclonal antibody.
 12. Thekit of claim 5 where said antibody is a polyclonal antibody.